(12) INTERNATIONAL APPLICAtlC>y^UBLISHED UNDER THE PATENT COOPERATION TREATY (PCT)
(19) World Intellectual Property
Organization
Internationa] Bureau
(43) International Publication Date
23 June 2005 (23.06.2005)
PCT
(10) International Publication Number
WO 2005/056759 A2
(51) International Patent Classification 7 :
C12N
(21) International Application Number:
PCTAJS2004/040694
(22) International Filing Date: 3 December 2004 (03.12.2004)
(25) Filing Language:
(26) Publication Language:
English
English
(30) Priority Data:
60/527,167
60/581,613
60/601,665
60/619,483
4 December 2003 (04. 12.2003) US
21 June 2004 (21.06.2004) US
13 August 2004 (13.08.2004) US
1 6 October 2004 (16.1 0.2004) US
(71) Applicant (for all designated States except US): XEN-
COR,INC. [US/US]; 111 W. Lemon Avenue, Monrovia,
CA 91016 (US).
(72) Inventors; and
(75) Inventors/Applicants (for US only): LAZAR, Gregory,
Alan [US/US]; Apt. C, 839 E. Mobeck Street, West Cov-
ina, CA 91790 (US). DESJARLAIS, John R. [US/US];
2096 E. Crary Street, Pasadena, CA 91104 (US). HAM-
MOND, Philip W. [US/US]; 421 Crestvale Drive, Sierra
Madre, CA 91024 (US).
(74) Agents: BOOTH, Paul, M. et ah; Heller Ehrman White &
McAuliffe LLP, Suite 300, 1666 K Street, NW, Washing-
ton, DC 20006- 1 228 (US).
(81) Designated States (unless otherwise indicated, for every
kind of national protection available): AE, AG, AL, AM,
AT, AU, AZ, B A, BB, BG, BR, BW, BY, BZ, CA, CH, CN,
CO, CR, CU, CZ, DE, DK, DM, DZ, EC, EE, EG, ES, FI,
GB, GD, GE, GH, GM, HR, HU, ID, 1L, IN, IS, JP, KE,
KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MA, MD,
MG, MK, MN, MW, MX, MZ, NA, Nl, NO, NZ, OM, PG,
PH, PL, PT, RO, RU, SC, SD, SE, SG, SK, SL, SY, TJ, TM,
TN, TR, TT, TZ, UA, UG, US, UZ, VC, VN, YU, ZA, ZM,
ZW.
(84) Designated States (unless otherwise indicated, for every
kind of regional protection available): ARIPO (BW, GH,
GM, KE, LS, MW, MZ, NA, SD, SL, SZ, TZ, UG, ZM,
ZW), Eurasian (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM),
European (AT, BE, BG, CH, CY, CZ, DE, DK, EE, ES, FI,
FR, GB, GR, HU, IE, IS, IT, LT, LU, MC, NL, PL, PT, RO,
[Continued on next page]
(54) Title: METHODS OF GENERATING VARIANT PROTEINS WITH INCREASED HOST STRING CONTENT AND COM-
POSITIONS THEREOF
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WO 2005/056759 A2 IIIllllllllfllHIIIlllllllllI
SE, SI, SK, TR), OAPI (BF, BJ, CF, CG, CI, CM, GA, GN, For two-letter codes and other abbreviations, refer to the "Guid-
GQ, GW, ML, MR, NE, SN, TD, TG). ance Notes on Codes and Abbreviations" appearing at the begin-
Published: nins °f each regular issue of the PCT Gazette.
— without international search report and to be republished
upon receipt of that report
WO 2005/056759
PCT/US2004/040694
METHODS OF GENERATING VARIANT PROTEINS WITH INCREASED HOST STRING
CONTENT AND COMPOSITIONS THEREOF
[01] This application claims the benefit of under 35 U.S.C. § 1 19(e) to USSNs
60/527,167, filed December 4, 2003; 60/581,613, filed June 21, 2004; 60/601,665, filed
August 13, 2004; and, 60/619,483, filed October 14, 2004; all of which are expressly
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[02] The present invention relates to novel methods for generating variant proteins with
increased host string content, and proteins that are engineered using these methods.
BACKGROUND OF THE INVENTION
[03] Many proteins that have the potential to be useful human therapeutics have a
xenogeneic origin. The use of xenogeneic proteins for therapeutic purposes may be
advantageous for a variety of reasons, including, for example, the established success of
hybridoma technology for raising antibodies in rodents, and the possibility of higher efficacy
with a xenogeneic protein than with a human counterpart. Although xenogeneic proteins are
a rich source of potential therapeutic molecules, they remain a relatively untapped one. One
reason for this is that nonhuman proteins are often immunogenic when administered to
humans, thereby greatly reducing their therapeutic utility. Additionally, even engineered
proteins of human origin may become immunogenic due to changes in the protein sequence.
[04] Immunogenicity is the result of a complex series of responses to a substance that is
perceived as foreign, and may include production of neutralizing and non-neutralizing
antibodies, formation of immune complexes, complement activation, mast cell activation,
inflammation, hypersensitivity responses, and anaphylaxis. Several factors can contribute to
protein immunogenicity, including but not limited to protein sequence, route and frequency of
administration, and patient population. Immunogenicity may limit the efficacy and safety of a
protein therapeutic in multiple ways. Efficacy can be reduced directly by the formation of
neutralizing antibodies. Efficacy may also be reduced indirectly, as binding to either
neutralizing or non-neutralizing antibodies typically leads to rapid clearance from serum.
Severe side effects and even death may occur when an immune reaction is raised. One
special class of side effects results when neutralizing antibodies cross-react with an
endogenous protein and block its function.
[05] Because of the clinical success of monoclonal antibodies, immunogenicity reduction
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of these proteins has been an intense area of investigation. Antibodies are a unique system
for the development of immunogenicity reduction methods because of the large number of
highly conserved antibody sequences and the Wealth of high-resolution structural
information. A number of strategies for reducing antibody immunogenicity have been
developed. The central aim of all of these approaches has been the reduction of nonhuman,
and correspondingly immunogenic content, while maintaining affinity for the antigen.
[06] The dominant method in use for antibody immunogenicity reduction, referred to as
"humanization", relies principally on the grafting of "donor" (typically mouse or rat)
complementarity determining regions (CDRs) onto "acceptor" (human) variable light chain
(VL) and variable heavy chain (VH) frameworks (FRs) (Tsurushita & Vasquez, 2004,
Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545, Elsevier
Science (USA)). This strategy is referred to as "CDR grafting" (Winter US 5225539).
"Backmutation " of selected acceptor framework residues to the corresponding donor
residues is often required to regain affinity that is lost in the initial grafted construct (US
5530101; US 5585089; US 5693761; US 5693762; US 6180370; US 5859205; US 5821337;
US 6054297; US 6407213). Despite the significant clinical application of antibodies
engineered using these methods, these methods remain nonrobust with regard to their
ability to reduce immunogenicity. A number of humanized antibodies have elicited
substantial immune reaction in clinical studies, with incidences of immune response as high
as 63 % of patients (Ritter et a/., 2001, Cancer Research 61: 6851-6859).
[07] The incomplete capacity of current humanization methods for immunogenicity
reduction are due to significant limitations imposed by the donor-acceptor approach.
Historically, the use of a single donor has been part of methods aimed at engineering a
single xenogeneic antibody to be suitable as a human biotherapeutic. However, the use of a
single acceptor is not required. On the contrary, the use of an acceptor antibody, and the
use of global homology to select it, place substantial restrictions on the immunogenicity
reduction process. A principal problem is that the use of overall sequence similarity between
nonhuman and human sequences as a metric for human immunogenicity is fundamentally
flawed. This means of measuring the degree of humanness does not accurately account for
the underlying molecular mechanisms of immune response. The immune system does not
recognize antigens on the basis of global sequence similarity to human proteins. Rather,
immune cells, including antigen presenting cells (APCs), T cells, and B cells, recognize
linear or conformational motifs comprising only a handful of residues. A key step in antigen
recognition is the formation of peptide-MHC-T cell receptor complexes. APCs express MHC
molecules that recognize short (approximately nine residue) linear peptide sequences,
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WO 2005/056759
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referred to as MHC agretopes. T cells express T cell receptors that recognize T cell
epitopes in the context of peptide-MHC complexes. T cells that recognize MHC agretopes
that are present in human proteins typically undergo apoptosis or become anergic, while T
cells that recognize foreign agretopes bound to MHC molecules may participate in an
immune response. Thus the relevant quantity for the immunogenicity of a protein is not its
global sequence similarity to a human sequence, but rather its sequence content of
individual human epitopes.
[08] The donor-acceptor model and the use of global sequence homology that it imposes
fails in practice. Because CDRs are treated as inviolable, structural incompatibilities are
introduced at the CDR-FR boundaries. Grafting of foreign donor CDRs onto a human
acceptor framework creates a substantial number of nonhuman epitopes in each variable
chain, including not only the epitopes in the foreign CDRs, but also the large number of
epitopes at the FR-CDR boundaries. This FR-CDR incompatibility is evident when one
backs away from global homology and looks at more local sequence homologies. CDR
grafting generally maximizes the donor-acceptor homology of the frameworks at the expense
of the CDRs (Clark, 2000, Immunology Today 21: 397-402). Ironically this frequently results
in lower global homology to human antibodies. In reality, the "cut and paste" approach to
imparting the functional determinants of a nonhuman antibody onto the framework of a
human one is unnecessary, as careful analysis of the antigen binding determinants of
antibodies shows that, in fact, the majority of CDR residues are not involved in binding
antigen (MacCallum et a/., 1996, J. Mol. Biol. 262: 732-745). FR-CDR incompatibility causes
not only immunological problems at the sequence level, but also causes conformational
problems at the structural level. As a result, humanization methods based on CDR grafting
often result in antigen affinity losses of 10-100 -fold, necessitating backmutation to donor
residues within the framework. This process of backmutation is a hallmark of essentially all
current humanization efforts, and because it introduces yet additional nonhuman epitopes,
highlights the inefficiency of these methods;.
[09] Methods that take an immune epitope approach to reducing antibody immunogenicity
have been explored (US 5712120; US 2003/0153043). Central to these methods is the
determination of sequences within a xenogeneic antibody that are in fact immunogenic
epitopes. Different methods for determination of immunogenicity both theoretical and
experimental have been described and include determination of potential for amphipathic
helix formation, binding to MHC, reactivity in a T-cell activation assay. A distinguishing
feature between these strategies and the present invention is that the present invention
makes no presumption as to the immunogenicity of specific epitopes. Rather, the primary
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goal is to maximize the content of human linear sequence strings in the xenogeneic antibody
as determined by comparison to an alignment of human sequences. The relevant sequence
dataset comprises strings that are nonimmunogenic for all relevant reasons, including lack of
interaction with MHC, lack of interaction with T cell receptor, lack of proper processing
necessary for presentation, and tolerance.
[10] It is noted that the methods described in US 5712120 and US 2003/0153043 suffer
additionally in that they fail to address a significant concern for local level sequence
engineering, namely the requirement for maintaining protein structure, stability, solubility,
and function. Thus, although the sequence string approach to immunogenicity reduction is
more accurate than CDR grafting, it will be optimal when coupled with protein design
methodology that takes into account both local sequence content and conformational
compatibility at the local and global structural level. In addition to providing scoring functions
for assessing host string content, the present invention also describes scoring functions that
evaluate other relevant properties of a protein that may be employed for the simultaneous
immunogenicity reduction and structural and functional optimization of proteins.
[111 In summary, the donor-acceptor model imposes significant restrictions on the
immunogenicity reduction process. With regard to sequence, global sequence homology is
an inappropriate metric for immunogenicity. With regard to structure, backmutations are
needed to repair conformational incompatibilities, thereby creating or reintroducing
nonhuman epitopes. The present invention describes a novel method for antibody
immunogenicity reduction that steps outside of the donor-acceptor model, and thus the
sequence and structural restrictions it imposes. The central strategy of the described
method is that it maximizes the content of human linear sequence strings. In this way
immunogenicity is addressed at the local sequence level, typically by utilizing the local
sequence information contained in an alignment of human sequences. This strategy not
only provides a more accurate measure of the immunogenicity, it enables substitutions to be
designed in a forward rather than backward manner to repair problems introduced by the
graft In effect, by addressing immunogenicity at the local sequence string level, the optimal
balance between binding determinants and humanness can be designed.
[12] The present invention describes a novel method for reducing the immunogenicity of
proteins that leverages the nonimmunogenic information contained in natural human
sequences to score protein sequences for immunogenic content at the sequence string
level. Furthermore, the described method capitalizes on recent advances in computational
sequence and structure-based protein engineering methods to quantitatively and
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systematically determine the optimal balance between human sequence content and protein
functionality. Because of the wealth of human sequence information available for the
immunoglobulin protein family, application to human antibodies is emphasized. Applications
to other proteins are also possible.
SUMMARY OF THE INVENTION
[13] The invention disclosed herein provides a novel method for reducing the
immunogenicity of a protein, wherein the method maximizes the content of sequence strings.
In a preferred embodiment, the method of the present invention maximizes the content of
human sequence strings.
[14] It is an object of the present invention to provide scoring functions that may be used
to evaluate the human sequence string content of a protein. In a preferred embodiment, the
scoring function compares the similarity of strings In a protein sequence to the strings that
compose a set of natural protein sequences. In another preferred embodiment, the set of
sequences is an aligned set of germline sequences. In additional preferred embodiments,
the set of sequences contains mature sequences. In the most preferred embodiments, the
sequences are human sequences.
[15] It is an object of the present invention to provide scoring functions that may be used
to evaluate the structural and/or functional fitness of a protein.
[16] It is an object of the present invention to provide protein variants of a parent protein
that are engineered using the methods described herein. In a preferred embodiment, the
parent protein is an immunoglobulin.
[17] It is an object of the present invention to provide experimental methods for screening
and testing the protein variants of the present invention.
[18] The present invention provides isolated nucleic acids encoding the protein variants
described herein. The present invention provides vectors comprising the nucleic acids,
optionally, operably linked to control sequences. The present invention provides host cells
containing the vectors, and methods for producing and optionally recovering the protein
variants.
[19] The present invention provides compositions comprising the protein variants
described herein, and a physiologically or pharmaceutical^ acceptable carrier or diluent.
[20] The present invention provides novel antibodies and Fc fusions that comprise the
protein variants disclosed herein. The novel antibodies and Fc fusions may find use in a
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therapeutic product.
[21] The present invention provides therapeutic treatment and diagnostic uses for the
protein variants disclosed herein.
BRIEF DESCRIPTION OF THE DRAWINGS
[22] Figure 1 . Human germ line sequences and diversity. The sequences that are known
to encode the human VH chains (Figure 1a), human VL kappa chains (Figure 1b), and VH
and VL kappa J chains (Figure 1c) are shown. The VL lambda germline sequences are not
provided. The germline sequences are numbered according to the numbering scheme of
Kabat (Kabat et a/., 1991 , Sequences of Proteins of Immunological Interest, 5th Ed., United
States Public Health Service, National Institutes of Health, Bethesda). The regions of the
variable region are indicated above the numbering in Figures 1a and 1b, and these include
framework regions 1 through 3 and the CDRs 1 through 3. Positions that make up the Kabat
CDRs are underlined. The germline chains are grouped into 7 subfamilies for V H and and 6
subfamilies for V L , as is known in the art, and these subfamilies are grouped together and
separated by a blank line. The sequences of the five germlines that make up the IgG light
kappa J chains (IGKJ1 - IGKJ5), and the six germlines that make up the IgG heavy J chains
(IGHJ1 - IGHJ5) are shown in Figure 1c. The kappa and lambda light J chains combine
with the VL/c and Vbl germlines respectively to form the light chain variable region, and the
heavy J chains combine with the VH germlines and heavy diversity (D) germlines (not
shown) to form the heavy chain variable region. The V H CDR3 is not part of the V H germ
line, and is encoded by the D and J genes.
[23] Figures 2. The quantities described by equations 1, 2, and 3 are illustrated. In
Figure 2a, IDstring (Equation 1) is illustrated for the string beginning at position i = 15,
comparing a region of the murine antibody m4D5 VH sequence (VH_m4D5) as parent
sequence s with the homologous region from the VH human germline sequence (VH_1-2) as
human sequence h. Only 30 residues from each sequence are shown, and the residues that
compose the relevant string are bolded. In Figure 2b, IDmax (Equation 2) is illustrated for
the parent sequence s string that begins at position i = 15 (shown in bold) and the
homologous regions from an aligned set of 7 VH human germline sequences. In Figure 2c,
HSC(s) (Equation 3) is illustrated for all strings (i=1 to i=22) in the parent sequence s and the
homologous regions from an aligned set of 7 VH human germline sequences.
[24] Figure 3. Sequence, host string content, and structure of WT AC 10 VL. Figure 3a
shows the sequence of the WT AC10 VL. Figure 3b shows the identity of each residue in
WT AC 10 VL as compared to the corresponding residue in each sequence of the human
6
WO 2005/056759
PCTAJS2004/040694
VUc germline. The black horizontal lines delineate the 7 different subfamilies as presented
in Figure 1, and the black vertical lines delineate the different framework and CDR regions of
the domain (in the order FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4). A grey square indicates
that the germline sequence has the same amino acid identity to the residue at the
corresponding position in the WT AC10 VL sequence. A white square indicates that the two
sequences differ at that position. Figure 3c shows the continuous 8- and 9-mer strings
between WT AC 10 VL and each sequence of the human VL/c germline. The black horizontal
and vertical lines are as described in Figure 3b. A grey square indicates that the germline
sequence comprises an 9-mer string centered on that position that is an 8 out of 9 or 9 out of
9 identical match to the corresponding string (centered on the corresponding residue) in the
WT AC10 VL sequence. Figure 3d shows the structure of the modeled WT AC10 variable
region. The light chain is shown as grey ribbon, the heavy chain is shown as black ribbon,
and the CDR residues are indicated as black lines.
[25] Figure 4. Sequence, host string content, and structure of WT AC10 VH. The figure
is as described in the figure legend for Figure 3, except that here the light chain is shown as
black ribbon and the heavy chain is shown as grey ribbon.
[26] Figure 5. Sequence, host string content, and structure of CDR grafted AC10 VL.
CDR grafted AC10 VL was derived from the CDRs of WT AC10 and the frameworks of the
human germline sequence vlk_4-1. Differences between CDR grafted AC10 VL and WT
AC10 VL are shown as bolded residues in the sequence in Figure 5a, and as black ball and
sticks in Figure 5d.
[27] Figure 6. Sequence, host string content, and structure of CDR grafted AC10 VH.
CDR grafted AC10 VH was derived from the CDRs of WT AC10 and the frameworks of the
human germline sequence vh_1-3 and substitutions Q108L and A1 13S (Kabat numbering) in
FR4.
[28] Figure 7. AC10 VL and VH variants with optimized HSC. AC10 VL and VH variants
with optimized HSC. The nonredundant set of output sequences from the calculations
described in Example 1 are shown. For each iteration (Iter) the following are provided: the
Structural Consensus; Structural Precedence; Human String Content (HSC); Human String
Similarity (HSS); N 9 max; the Framework Region Homogeneity (FRH); and, the number of
mutations from WT (Muts). The output sequences were clustered based on their mutational
distance from the other sequences in the set. These clusters are delineated by the
horizontal black lines. The "Cluster" column provides this mutational distance quantitatively.
Differences between the parent WT AC 10 sequence are shown in grey. Positions are
7
WO 2005/056759 PCT/US2004/040694
numbered according to the Kabat numbering scheme, provided at the top. The light grey
regions bracketed by the black horizontal lines indicate residues in or proximal to the Kabat
defined CDRs that were masked in the calculation. Sequence differences from WT C225 VL
are shown in dark grey.
[29] Figure 8. Sequence, host string content, and structure of L1 AC1 0 VL.
[30] Figure 9. Sequence, host string content, and structure of L2 AC10 VL.
[31] Figure 1 0. Sequence, host string content, and structure of L3 AC10 VL.
[32] Figure 11 . Sequence, host string content, and structure of H1 AC10 VH.
[33] Figure 1 2. Sequence, host string content, and structure of H2 AC1 0 VH.
[34] Figure 1 3. Sequence, host string content, and structure of H3 AC1 0 VH.
[35] Figures 14. AlphaScreen™ assay measuring binding between AC10 variants and
the target antigen CD30. In the presence of competitor variant antibody, a characteristic
inhibition curve is observed as a decrease in luminescence signal. The binding data were
normalized to the maximum and minimum luminescence signal for each particular curve,
provided by the baselines at low and high antibody concentrations respectively. The curves
represent the fits of the data to a one site competition model using nonlinear regression, and
the fits provide IC50s for each antibody.
[36] Figure 15. Figure 1 1 . SPR sensorgrams showing binding of AC10 WT and variant
full length antibodies to the CD30 target antigen. The curves consist of an association
phase and dissociation phase, the separation being marked by a little spike on each curve.
[37] Figure 16. AlphaScreen™ assay measuring binding between AC10 variants and
human V1 58 FcKRHIa.
[38] Figure 17. Cell-based ADCC assay of WT and AC10 variants. Purified human
peripheral blood monocytes (PBMCs) were used as effector cells, L540 Hodgkin's
lymphoma cells were used as target cells, and lysis was monitored by measuring LDH
activity using the Cytotoxicity Detection Kit (LDH, Roche Diagnostic Corporation,
Indianapolis, IN ). Samples were run in triplicate to provide error estimates (n=3, +/- S.D.).
Figure 17 shows the dose dependence of ADCC at various antibody concentrations, and the
curves represent the fits of the data to a sigmoidal dose-response model using nonlinear
regression. Raw data are presented in Figures 17a and 17b, whereas in Figure 17c the data
were normalized to a percentage scale of maximal cytotoxicity determined by Triton-X100
lysis of target cells.
8
WO 2005/056759
PCT/US2004/040694
[39] Figure 18. Cell-based assay measuring ADCC capacity of WT (H0/L0) and H3/L3
AC10 antibodies comprising Fc variants that provide enhanced effector function. Raw data
were normalized to a percentage scale of maximal cytotoxicity determined by Triton-X100
lysis of target cells.
[40] Figure 19. AlphaScreen™ assay measuring binding between select H3L3 secondary
AC10 variants and the target antigen CD30.
[41] Figure 20. Sequence, host string content, and structure of L3.71 AC10 VL.
[42] Figure 21 . Sequence, host string content, and structure of L3.72 AC10 VL
[43] Figure 22. Sequence, host string content, and structure of H3.68AC10VH.
[44] Figure 23. Sequence, host string content, and structure of H3.69 AC10 VH.
[45] Figure 24. Sequence, host string content, and structure of H3.70 AC10 VH.
[46] Figure 25. Amino acid sequences of a AC10 variant antibodies comprising the L3.71
AC10 variant VL with the CLD constant light chain (Figure 25a) and the H3.70 AC10 variant
VH with IgG constant chains (Figures 25b - 25e) that may comprise amino acid
modifications in the Fc region. Figure 25b provides an lgG1 heavy chain with positions that
may be mutated designated in bold as X 1t X 2 , X 3 , and X4, referring to residues S239, V264,
A330, and I332. Figure 25c provides one example of a heavy chain described in Figure 25b,
here comprising the H3.70 AC10 variant VH region with the S239D/A330L/I332E lgG1
constant region. Figure 25d provides an lgG2 heavy chain with positions that may be
mutated and designated in bold as X 1t X 2 , X 3 , X4, Z 1f Z 2 , Z3, Z4, and Z 5 referring to residues
S239, V264, A330, 1332, P233, V234, A235, -236, and G237 (here -236 refers to a deletion
at EU index position 236). Figure 25e provides one example of a heavy chain described in
Figure 25d, here comprising the H3.70 AC10 variant VH region with the
S239D/A330L/I332E/P233E/V234L/A235L/-236G lgG2 constant region. .
[47] Figure 26. Sequence, host string content, and structure of WT C225 VL.
[48] Figure 27. Sequence, host string content, and structure of WT C225 VH.
[49] Figure 28. Sequence, host string content, and structure of CDR grafted C225 VL,
which was derived from the CDRs of WT C225 and the frameworks of the human germline
sequence vik_6D-21 and an L106I (Kabat numbering) substitution in FR4.
[50] Figure 29. Sequence, host string content, and structure of CDR grafted C225 VH,
which was derived from the CDRs of WT C225 and the frameworks of the human germline
sequence vh_4-30-4 and an A1 13S (Kabat numbering) substitution in FR4.
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[51] Figure 30. C225 VL and VH variants with optimized HSC. The nonredundant set of
output sequences from the calculations described in Example 2 are shown.
[52] Figure 31 . C225 VL and VH variants with optimized HSC. The nonredundant set of
output sequences from the calculations described in Example 2 are shown.
[53] Figure 32. Sequence, host string content, and structure of 12 C225 VL.
[54] Figure 33. Sequence, host string content, and structure of L3 C225 VL.
[55] Figure 34. Sequence, host string content, and structure of L4 C225 VL.
[56] Figure 35. Sequence, host string content, and structure of H3 C225 VH.
[57] Figure 36. Sequence, host string content, and structure of H4 C225 VH.
[58] Figure 37. Sequence, host string content, and structure of H5 C225 VH.
[59] Figure 38. Sequence, host string content, and structure of H6 C225 VH.
[60] Figure 39. Sequence, host string content, and structure of H7 C225 VH.
[61] Figure 40. Sequence, host string content, and structure of H8 C225 VH.
[62] Figure 41 , SPR sensorgrams showing binding of full length antibody C225 variants
to the EGFR target antigen. The sensorgrams show binding of C225 WT (L0/H0) and
variant (L0/H3, L0/H4, L0/H5, L0/H6, L0/H7, L0/H8, L2/H3, L2/H4, L2/H5, L2/H6, L2/H7,
L2/H8, L3/H3, L3/H4, L3/H5, L3/H6, L3/H7, L3/H8, L4/H3, L4/H4, L4/H5, L4/H6, L4/H7, and
L4/H8)full length antibodies to the EGFR sensor chip. The curves consist of an association
phase and dissociation phase, the separation being marked by a little spike on each curve.
[63] Figures 42. Cell-based ADCC assay of C225 WT (L0/H0) and variant (L0/H3, L0/H4,
L0/H5, L0/H6, L0/H7, L0/H8, L2/H3, L2/H4, L2/H5, L2/H6, L2/H7, L2/H8, L3/H3, L3/H4,
L3/H5, L3/H6, L3/H7, L3/H8, L4/H3, L4/H4, L4/H5, L4/H6, L4/H7, and L4/H8) full length
antibodies. Purified human peripheral blood monocytes (PBMCs) were used as effector
cells, A431 epidermoid carcinoma cells were used as target cells at a 10:1 effectontarget cell
ratio, and lysis was monitored by measuring LDH activity using the Cytotoxicity Detection Kit
(LDH, Roche Diagnostic Corporation, Indianapolis, IN ). Samples were run in triplicate to
provide error estimates (n=3, +/- S.D.). Figure 42 shows the dose dependence of ADCC at
various antibody concentrations, normalized to the minimum and maximum levels of lysis for
the assay. The curves represent the fits of the data to a sigmoidal dose-response model
using nonlinear regression.
[64] Figure 43. Sequence, host string content, and structure of WT ICR62 VL.
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[65] Figure 44. Sequence, host string content, and structure of WT ICR62 VH.
[66] Figure 45. Sequence, host string content, and structure of CDR grafted ICR62 VL.
CDR grafted ICR62 VL was derived from the CDRs of WT ICR62 and the frameworks of the
human germline sequence vlk_1-17 and an L106I (Kabat numbering) substitution in FR4.
[67] Figure 46. Sequence, host string content, and structure of CDR grafted ICR62 VH.
CDR grafted ICR62 VH was derived from the CDRs of WT ICR62 and the frameworks of the
human germline sequence vhjl-f and substitutions A107T and S108L (Kabat numbering) in
FR4.
[68] Figure 47. ICR62 VL and VH variants with optimized HSC.
[69] Figure 48. Sequence, host string content, and structure of L3 ICR62 VL.
[70] Figure 49. Sequence, host string content, and structure of H9 ICR62 VH.
[71] Figure 50. Sequence, host string content, and structure of H10 ICR62 VH.
[72] Figure 51 . Comparison of VH sequences humanized by the methods in the prior art
versus the present method. Prior art antibodies include Ctm01 , A5B7, Zenapax, MaE1 1 f
1129, MHM2, H52, Huzaf, Hu3S193, D3H44, AQC2, 2C4, D3H44, Hfe7A, 5C8, m4D5,
A.4.6.1, Campath, HuLys11, A.4.6.1, Mylotarg, MEDI-507, huH65_vh, EP-5C7, 9F3, HPC4,
38C2, Br96, 1A6, and 6.7. Sequences designed using the present invention, including AC10
H1, H2, and H3 f C225 H3, H4, H5, H6 f H7 ? and H8, and ICR62 H9 and H10, are offset to the
right. Figure 51a provides the host string content (HSC) as defined by equation 3, Figure
51b provides the exact string content (ESC) as defined by equation 3a, and Figure 51c
provides the framework region homogeneity (FRH) as defined by equation 10. Window size
w was 9 for all calculations.
[73] Figure 52. Comparison of VL sequences humanized by the methods in the prior art
versus the present method. Prior art antibodies include Ctm01 , A5B7, Zenapax, MaE1 1 ,
1129, MHM2, H52, Huzaf, Hu3S193, D3H44, AQC2, 2C4, D3H44, Hfe7A, 5C8, m4D5,
A.4.6.1, Campath, HuLys11, A.4.6.1, Mylotarg, MEDI-507, huH65_vh, EP-5C7, 9F3, HPC4,
38C2, Br96, 1 A6, and 6.7. Sequences designed using the present invention, including AC10
L1, L2, and L3, C225 L2, L3, L4, and ICR62 L2, are offset to the right. Figure 52a provides
the host string content (HSC) as defined by equation 3, Figure 52b provides the exact string
content (ESC) as defined by equation 3a, and Figure 52c provides the framework region
homogeneity (FRH) as defined by equation 10. Window size w was 9 for all calculations.
DETAILED DESCRIPTION OF THE INVENTION
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Definitions
[74] In order that the invention may be more completely understood, several definitions
are set forth below. Such definitions are meant to encompass grammatical equivalents.
[75] By " amino acid" as used herein is meant one of the 20 naturally occurring amino
acids or any non-natural analogues that may be present at a specific, defined position.
[76] By " amino acid modification " herein is meant an amino acid substitution, insertion,
and/or deletion in a polypeptide sequence. The preferred amino acid modification herein is a
substitution.
[77] By " amino acid substitution " or "substitution" herein is meant the replacement of an
amino acid at a given position in a protein sequence with another amino acid.
[78] By " antibody " herein is meant a protein consisting of one or more proteins
substantially encoded by all or part of the recognized immunoglobulin genes. The
recognized immunoglobulin genes, for example in humans, include the kappa (/c), lambda
(/I), and heavy chain genetic loci, which together comprise the myriad variable region genes,
and the constant region genes mu (p), delta (6), gamma (y) 9 sigma (a), and alpha {a) which
encode the IgM, IgD, IgG, IgE, and IgA isotypes respectively. Antibody herein is meant to
include full length antibodies and antibody fragments, and may refer to a natural antibody
from any organism, an engineered antibody, or an antibody generated recombinantly for
experimental, therapeutic, or other purposes. By " IgG " as used herein is meant a protein
belonging to the class of antibodies that are substantially encoded by a recognized
immunoglobulin gamma gene. In humans this class comprises IgGt, lgG2, lgG3, and lgG4.
[79] By " corresponding " or " eguivalenf residues as meant herein are residues that
represent similar or homologous sequence and/or structural environments between a first
and second protein, or between a first protein and set of multiple proteins. In order to
establish homology, the amino acid sequence of a first protein is directly compared to the
sequence of a second protein. After aligning the sequences, using one or more of the
homology alignment programs known in the art (for example using conserved residues as
between species), allowing for necessary insertions and deletions in order to maintain
alignment (i.e., avoiding the elimination of conserved residues through arbitrary deletion and
insertion), the residues equivalent to particular amino acids in the primary sequence of the
first protein are defined. Alignment of conserved residues preferably should conserve 100%
of such residues. However, alignment of greater than 75% or as little as 50% of conserved
residues is also adequate to define equivalent residues. Corresponding residues may also
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be defined by determining structural homology between a first and second protein that is at
the level of tertiary structure for proteins whose structures have been determined. In this
case, equivalent residues are defined as those for which the atomic coordinates of two or
more of the main chain atoms of a particular amino acid residue of the proteins (N on N, CA
on CA, C on C and O on O) are within 0.13 nm and preferably 0.1 nm of each other after
alignment. Alignment is achieved after the best model has been oriented and positioned to
give the maximum overlap of atomic coordinates of non-hydrogen protein atoms of the
proteins.
[80] By "CDR " as used herein is meant a Complementarity Determining Region of an
antibody variable domain. Systematic identification of residues included in the CDRs have
been developed by Kabat (Kabat et ah, 1991 , Sequences of Proteins of Immunological
Interest, 5th Ed., United States Public Health Service, National Institutes of Health,
Bethesda) and alternately by Chothia (Chothia & Lesk, 1987, J. Mol. Biol. 196: 901-917;
Chothia et al., 1989, Nature 342: 877-883; Al-Lazikani et al., 1997, J. MoL BioL 273: 927-
948). For the purposes of the present invention, CDRs are defined as a slightly smaller set
of residues than the CDRs defined by Chothia. VL CDRs are herein defined to include
residues at positions 27-32 (CDR1), 50-56 (CDR2), and 91-97 (CDR3), wherein the
numbering is according to Chothia. Because the VL CDRs as defined by Chothia and Kabat
are identical, the numbering of these VL CDR positions is also according to Kabat. VH
CDRs are herein defined to include residues at positions 27-33 (CDR1), 52-56 (CDR2), and
95-102 (CDR3), wherein the numbering is according to Chothia. These VH CDR positions
correspond to Kabat positions 27-35 (CDR1 ), 52-56 (CDR2), and 95-1 02 (CDR3).
[81] By " framework" as used herein is meant the region of an antibody variable domain
exclusive of those regions defined as CDR's. Each antibody variable domain framework can
be further subdivided into the contiguous regions separated by the CDR's (FR1, FR2, FR3
and FR4).
[82] By "germline" as used herein is meant the set of sequences that compose the natural
genetic repertoire of a protein, and its associated alleles.
[83] , By " host" as used herein is meant a family, genus, species or subspecies, group of
individuals or even a single individual. A host group of individuals can be selected for based
upon a variety of criteria, such as MHC allele composition, etc. In a preferred embodiment,
a host is canine, murine, primate, or human. In the most preferred embodiment, a host is
human.
[84] By "host string " or " host sequence 0 as used herein is meant a string or sequence that
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encodes any part of a naturally occurring host protein.
[85] By " humanized " antibody as used herein is meant an antibody comprising a human
framework region and one or more CDR's from a non-human (usually mouse or rat)
antibody. The non-human antibody providing the CDR's is called the " donor and the human
immunoglobulin providing the framework is called the " acceptor" . One says that the donor
antibody has been humanized", by the process of "humanization".
[86] By " identity" as used herein is meant the number of residues in a first sequence that
are identical to the residues in a second sequence after alignment of the sequences to
achieve the maximum identity.
[87] By " immune epitope " or " epitope " herein is meant a linear sequence of amino acids
that is located in a protein of interest. Epitopes may be analyzed for their potential for
immunogenicity. Epitopes may be any length, preferably 9-mers.
[88] By "immunogenicity" herein is meant the ability of a protein to elicit an immune
response, including but not limited to production of neutralizing and non-neutralizing
antibodies, formation of immune complexes, complement activation, mast cell activation,
inflammation, and anaphylaxis.
[89] By " immunoglobulin do) " herein is meant a protein consisting of one or more proteins
substantially encoded by immunoglobulin genes. Immunoglobulins include but are not
limited to antibodies. Immunoglobulins may have a number of structural forms, including but
not limited to full length antibodies, antibody fragments, and individual immunoglobulin
domains. By " immunoglobulin (lg) domain " herein is meant a region of an immunoglobulin
that exists as a distinct structural entity as ascertained by one skilled in the art of protein
structure. Ig domains typically have a characteristic p-sandwich folding topology. The
known Ig domains in the IgG class of antibodies are V H , CH1 (Cy1), CH2 (Cy2), CH3 (Cy3),
V Ll and C L .
[90] By "natural seouence " or " natural protein" as used herein is meant a protein that has
been determined to exist absent any experimental modifications. Also included are
sequences that can be predicted to exist in nature based on experimentally determined
sequences. An example of such a predicted sequence is an antibody that can be predicted
to exist based on the established patterns of germline recombination! In this case the large
size of the predicted antibody repertoire makes the actual experimental determination of all
mature recombined antibodies not practical.
[91] By " parent " or " parent protein " as used herein is meant a protein that is subsequently
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modified to generate a variant. The parent protein may be a naturally occurring protein, or a
variant or engineered version of a naturally occurring protein. Parent protein may refer to
the protein itself, compositions that comprise the parent protein, or the amino acid sequence
that encodes it Accordingly, by " parent antibody" as used herein is meant an antibody that
is subsequently modified to generate a variant antibody. Accordingly, by " parent sequence"
as used herein is meant the sequence that encodes the parent protein or parent antibody.
[92] By " position " as used herein is meant a location in the sequence of a protein.
Positions may be numbered sequentially, or according to an established format, for example
Kabat, Chothia, and/or the EU index as in Kabat.
[93] By "protein" herein is meant at least two covalently attached amino acids, which
includes proteins, polypeptides, oligopeptides and peptides. The protein may be made up of
naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures.
[94] By " reduced immunogenicitv" herein is meant a decreased ability to activate the
immune system, when compared to the parent protein. For example, a protein variant can
be said to have "reduced immunogenicity" if it elicits neutralizing or non-neutralizing
antibodies in lower titer or in fewer patients than the parent protein. A protein variant also
can be said to have "reduced immunogenicity" if it shows decreased binding to one or more
MHC alleles or if it induces T cell activation in a decreased fraction of patients relative to the
parent protein.
[95] By " residue " as used herein is meant a position in a protein and its associated amino
acid identity. For example, proline 9 (also referred to as Pro9, also referred to as P9) is a
residue in the WT AC1 0 VH region.
[96] By " scoring function" herein is meant any equation or method for evaluating the
fitness of one or more amino acid modifications in a protein. The scoring function may
involve a physical or chemical energy term, or may involve knowledge-, statistical-,
sequence-based energy terms, and the like.
[97] By " string" as used herein is meant a contiguous sequence that encodes any part of
a protein. Strings may comprise any 2 or more linear residues, with the number of
contiguous residues being defined by the " window " or " window size ". Window sizes of 2 - 20
are preferred, with 7-13 more preferred, with 9 most preferred.
[98] By " target" as used herein is meant the molecule that is bound specifically by a
protein. A target may be a protein, carbohydrate, lipid, or other chemical compound. The
target of an antibody is its antigen, also referred to as its target antigen.
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[99] By "variable region" as used herein is meant the region of an immunoglobulin that
comprises one or more Ig domains substantially encoded by any of the VL (including V/c and
VA) and/or V H genes that make up the light chain (including kappa and lambda) and heavy
chain immunoglobulin genetic loci respectively. A light or heavy chain variable region (VL
and VH) consists of a "framework" or TFT region interrupted by three hypervariable regions
referred to as "complementarity determining regions" or "CDRs". The extent of the
framework region and CDRs have been precisely defined, for example as in Kabat (see
"Sequences of Proteins of Immunological Interest," E. Kabat et al., U.S. Department of
Health and Human Services, (1983)), and as in Chothia. The framework regions of an
antibody, that is the combined framework regions of the constituent light and heavy chains,
serves to position and align the CDRs, which are primarily responsible for binding to an
antigen.
[100] By "variant protein " or " protein variant " . or "variant " as used herein is meant a protein
that differs from a parent protein by virtue of at least one amino acid modification. Protein
variant may refer to the protein itself, a composition comprising the protein, or the amino
sequence that encodes it Preferably, the protein variant has at least one amino acid
modification compared to the parent protein, e.g. from about one to about ten amino acid
modifications, and preferably from about one to about five amino acid modifications
compared to the parent. The protein variant sequence herein will preferably possess at least
about 80% homology with a parent protein sequence, and most preferably at least about
90% homology, more preferably at least about 95% homology. Accordingly, by
" immunoglobulin variant " as used herein is meant an immunoglobulin that differs from a
parent immunoglobulin by virtue of at least one amino acid modification.
[101] By "wild type orWT" herein is meant an amino acid sequence or a nucleotide
sequence that is found in nature and includes allelic variations. A WT protein has an amino
acid sequence or a nucleotide sequence that has not been intentionally modified.
[102] The protein variants of the present invention may be derived from parent proteins
that are themselves from a wide range of sources. The parent protein may be substantially
encoded by one or more genes from any organism, including but not limited to humans,
mice, rats, rabbits, camels, llamas, dromedaries, monkeys, preferably mammals and most
preferably humans and mice and rats. Although in a preferred embodiment the parent
protein is nonhuman, in some embodiments of the present invention the parent protein may
be human or similar to human. The parent protein may comprise more than one protein
chain, and thus may be a monomer or an oligomer, including a homo- or hetero-oligomer. In
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a preferred embodiment, the parent protein is an antibody, referred to as the parent
antibody. The parent antibody need not be naturally occurring. For example, the parent
antibody may be an engineered antibody, including but not limited to nonhuman and
chimeric antibodies. The parent antibody may be fully human, obtained for example using
transgenic mice (Bruggemann et a/., 1997, Curr Opin Biotechnol 8:455-458) or human
antibody libraries coupled with selection methods (Griffiths et a/., 1998, Curr Opin Biotechnol
9:102-108). The parent antibody need not be naturally occurring. For example, the parent
antibody may be an engineered antibody, including but not limited to chimeric antibodies and
humanized antibodies (Clark, 2000, Immunol Today 21:397-402). The parent antibody may
be an engineered variant of an antibody that is substantially encoded by one or more natural
antibody genes. In one embodiment, the parent antibody has been affinity matured, as is
known in the art, or engineered in some other way. The parent antibodies of the present
invention may be substantially encoded by immunoglobulin genes belonging to any of the
antibody classes, and may comprise sequences belonging to the IgG class of antibodies,
including lgG1 , lgG2, lgG3, or lgG4, or alternatively the IgA (including subclasses lgA1 and
lgA2), IgD, IgE, IgG, or IgM classes of antibodies.
[103] Virtually any binding partner or antigen may be targeted by the proteins of the
present invention. A number biotherapeutic proteins and antibodies that are approved for
use, in clinical trials, or in development may thus benefit from immunogenicity reduction
methods of the present invention. In a preferred embodiment, the less immunogenic protein
of the present invention is an antibody. The less immunogenic antibody may comprise
sequences belonging to the IgG (including lgG1 , lgG2, lgG3, or lgG4), IgA (including
subclasses lgA1 and lgA2), IgD, IgE, IgG, or IgM classes of antibodies, with the IgG class
being preferred. The less immunogenic antibodies of the present invention may be full
length antibodies, or antibody fragments. Constant regions need not be present, but if they
are, they will likely be substantially identical to human immunoglobulin constant regions.
[104] The constant region of the antibody may be modified in some way to make it more
effective therapeutically. For example, the constant region may comprise substitutions that
enhance therapeutic properties. Most preferred substitutions and optimized effector function
properties are described in USSN 10/672,280, PCT US03/30249, and USSN 10/822,231,
and USSN 60/627,774, filed 1 1/12/2004 and entitled "Optimized Fc Variants". Other known
Fc variants that may find use in the present invention include but are not limited to those
described in US.6,737,056; PCT US2004/000643; USSN 10/370,749; PCT/US2004/005112;
US 2004/0132101; USSN 10/672,280; PCT/US03/30249; US 6,737,056, US 2004/0002587;
WO 2004/063351; Idusogie et al.,.2001, J. Immunology 166:2571-2572; Hinton et al., 2004,
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J. Biol. Chem. 279(8): 6213-6216. In alternate embodiments, the constant region may
comprise one or more engineered glycoforms, as is known in the art (Umana et a/., 1999,
Nat Biotechnol 17:176-180; Davies ef a/., 2001, Biotechnol Bioeng 74:288-294; Shields et
a/., 2002, J Biol Chem 277:26733-26740; Shinkawa et a/ M 2003, J Biol Chem 278:3466-
3473); (US 6,602,684; USSN 10/277,370; USSN 10/113,929; PCT WO 00/61739A1; PCT
WO 01/29246A1 ; PCT WO 02/31 140A1 ; PCT WO 02/30954A1); (Potelligent™ technology
[Biowa, Inc., Princeton, NJ]; GlycoMAb™ glycosylation engineering technology [GLYCART
biotechnology AG, Zurich, Switzerland]).
[105] The protein variants of the present invention may find use in a wide range of protein
products. In one embodiment the protein is a therapeutic, a diagnostic, or a research
reagent, preferably a therapeutic. Alternatively, the protein of the present invention may be
used for agricultural or industrial uses. In a preferred embodiment, the protein is a
therapeutic that is used to treat a disease. By "disease " herein is meant a disorder that may
be ameliorated by the administration of a pharmaceutical composition comprising a protein
of the present invention. Diseases include but are not limited to autoimmune diseases,
immunological diseases, infectious diseases, inflammatory diseases, neurological diseases,
and oncological and neoplastic diseases including cancer. In one embodiment, a protein of
the present invention is the only therapeutically active agent administered to a patient.
Alternatively, the protein of the present invention is administered in combination with one or
more other therapeutic agents, including but not limited to cytotoxic agents,
chemotherapeutic agents, cytokines, growth inhibitory agents, anti-hormonal agents, kinase
inhibitors, anti-angiogenic agents, cardioprotectants, or other therapeutic agents. The
proteins of the present invention may be combined with other therapeutic regimens. For
example, in one embodiment, the patient to be treated with the protein may also receive
radiation therapy and/or undergo surgery. In an alternate embodiment, the protein of the
present invention is conjugated or operably linked to another therapeutic compound. The
therapeutic compound may be a cytotoxic agent, a chemotherapeutic agent, a toxin, a
radioisotope, a cytokine, or other therapeutically active agent. In yet another embodiment, a
protein of the present invention may be conjugated to a protein or molecule for utilization in
tumor pretargeting or prodrug therapy. Other modifications of the proteins of the present
invention are contemplated herein. For example, the protein may be linked to one of a
variety of nonproteinaceous polymers, for example e.g., polyethylene glycol (PEG).
[106] Pharmaceutical compositions are contemplated wherein a protein of the present
invention and one or more therapeutically active agents are formulated. Formulations of the
proteins of the present invention are prepared for storage by mixing the protein having the
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desired degree of purity with optional pharmaceutical^ acceptable carriers, excipients or
stabilizers (Remingtons Pharmaceutical Sciences 16th edition, Osol, A. Ed.,1980), in the
form of lyophilized formulations or aqueous solutions. The formulations to be used for in
vivo administration are preferably sterile. The proteins disclosed herein may also be
formulated as immunolipospmes, or entrapped in microcapsules. The concentration of the
protein of the present invention in the formulation may vary from about 0.1 to 1 00 weight %.
In a preferred embodiment, the concentration of the protein is in the range of 0.003 to 1.0
molar. In order to treat a patient, a therapeutically effective dose of the protein of the
present invention may be administered. The exact dose will depend on the purpose of the
treatment, and will be ascertainable by one skilled in the art using known techniques.
Dosages may range from 0.01 to 100 mg/kg of body weight or greater, for example 0.1, 1,
10, or 50 mg/kg of body weight, with 1 to 10mg/kg being preferred. Administration of the
pharmaceutical composition comprising a protein of the present invention, preferably in the
form of a sterile aqueous solution, may be done in a variety of ways, including, but not
limited to, orally, subcutaneously, intravenously, intranasally, intraotically, transdermal^,
topically, intraperitoneally, intramuscularly, intrapulmonary, inhalably, vaginally, parenterally,
rectally, or intraocularly. As is known in the art, the pharmaceutical composition may be
formulated accordingly depending upon the manner of introduction.
Description of the Methodology
[107] The present invention provides a novel method for reducing the immunogenicity of a
protein. A central principle of the described method is that substitutions are designed to
maximize the content of human linear sequence strings using an alignment of human
sequences. For application to antibodies, this approach to immunogenicity reduction
excludes the use of the single donor-acceptor model employed in humanization methods.
By stepping outside of the limitations imposed by the need to choose a human acceptor
sequence a priori, a more immunologically relevant approach to immunogenicity reduction is
enabled. Sequence information and structural information may be used to score potential
amino acid substitutions. The scoring results are used to design protein variant libraries,
which are subsequently screened experimentally to determine favorable substitutions.
Feedback from experimental data may guide subsequent iterations of design and
experimental screening, ultimately enabling protein variants to be engineered with the
optimal balance between biophysical and immunological constraints.
Sequences
[108] Central to the method described herein is that a set of host sequences provides
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information as to the degree to which linear sequence strings have the potential to be
immunogenic. Thus the set of sequences employed is an important parameter. In the most
common embodiment, the sequences are a set of human sequences that are homologous in
sequence and/or structure to the parent sequence. As is known in the art, some proteins
share a common structural scaffold and are homologous in sequence. This information may
be used to gain insight into particular positions in the protein family. Sequence alignments
are often carried out to determine which protein residues are conserved and which are not
conserved. That is to say, by comparing and contrasting alignments of protein sequences,
the degree of variability at a position may be observed, and the types of amino acids that
occur naturally at positions may be observed. Thus for the present invention, typically the
sequences are aligned such that the conserved or similar residues that exist between the
parent sequence and the set of human sequences and among the set of human sequences
can be identified. Methods for sequence alignment are well known in the art, and include
alignments based on sequence and structural homology.
[109] Protein sequence information can be obtained, compiled, and/or generated from
sequence alignments of naturally occurring proteins from any organism, including but not
limited to mammals. Because a preferred embodiment of present invention is directed
towards immunogenicity reduction for biotherapeutics, the sequences that compose the set
are most preferably human. The source of the sequences may vary widely, may be a
database that is compiled publicly or privately, and may be may include one or more of the
known general protein and nucleic acid sequences databases, including but not limited to
SwissProt, GenBank and Entrez, and EMBL Nucleotide Sequence Database. Because a
preferred embodiment of the present invention is its application to the immunogenicity
reduction of immunoglobulins, a number of immunoglobulin databases may be useful for
obtaining sequences, including but not limited to the Kabat database (Johnson & Wu, 2001,
Nucleic Acids Res 29:205-206; Johnson & Wu, 2000, Nucleic Acids Res 28:214-218), the
IMGT database (IMGT, the international ImMunoGeneTics information system®; Lefranc et
a/., 1999, Nucleic Acids Res 27:209-212; Ruiz et a/., 2000 Nucleic Acids Re. 28:219-221;
Lefranc et a/., 2001 , Nucleic Acids Res 29:207-209; Lefranc et a/., 2003, Nucleic Acids Res
31 :307-31 0), and VBASE.
[110] As is well known in the art, immunoglobulins possess a high degree of sequence and
structural homology, and therefore alignment of sequences provides a wealth of information.
Due to the existence of deletions and insertions in these alignments, numbering conventions
have been adopted to enable a normalized reference to conserved positions in
immunoglobulin families or subfamilies. Those skilled in the art will appreciate that these
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conventions consist of nonsequential numbering in specific regions of an immunoglobulin
sequence, and thus accordingly the positions of any given immunoglobulin as defined by any
given numbering scheme will not necessarily correspond to its sequential sequence or to
those in an alternate numbering scheme. For all variable regions discussed in the present
invention, numbering is according to the numbering scheme of Kabat (Kabat et a/., 1991 ,
Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health
Service, National Institutes of Health, Bethesda). For all constant region positions discussed
in the present invention, number is according to the EU index as in Kabat Alternate
numbering schemes may find use in the present invention, including but not limited that of
Chothia (Chothia & Lesk, 1987, J.Mol. Biol 196: 901-917; Chothia et al t 1989, Nature 342:
877-883; Al-Lazikani et a/., 1997, J. Mol Biol 273: 927-948).
[111] In a most preferred embodiment, the set of human sequences used is an aligned set
of human germline immunoglobulin sequences. For example, Figures 1a - 1c provide the
set of sequences that compose the human antibody variable region germline (VH, VL, and J
chains), along with the corresponding diversity at each position. The human germline
repertoire for immunoglobulin heavy chain variable regions and immunoglobulin light chain
kappa variable regions have been reported (Matsuda et al., 1998, J Exp Med 188: 2151-
2162; Zachau, 2000, Biol Chem 381:951-954; Pallares et al., 1999, Exp Clin Immunogenet
16(1): 36-60; Barbie & Lefranc, 1998, Exp Clin Immunogenet 15(3): 171-83). The human
immunoglobulin kappa variable (IGKV) genes and joining (IGKJ) segments. Barbie V,
Lefranc MP). The rationale for use of this type of sequence information as a metric for
humanness is that the strings that compose the human germline should be minimally
immunogenic. Sequences need not be human genomic or germline sequences. In other
preferred embodiments, human antibody variable region sequences are derived not from
germline information, but rather from matured antibodies obtained for example from
hybridoma technology or cDNA libraries.
[112] For many of the genes in the human immunoglobulin germline, several different
alleles have been identified. Although the polymorphisms detected in many of the alleles do
not change the amino acid sequence of the gene, in a great number of cases the sequence
is changed. In choosing a set of sequences to use in the method described herein, different
sets of sequences may be chosen. When choosing a single allele as representative of a
specific gene the most cautious approach is to choose that sequence which is closest to the
consensus of the entire germline. This subset of sequences would thereby be most likely to
be represented within the population as a whole. Alternatively, a much greater sequence
diversity could be sampled by choosing representative sequences that are furthest from the
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consensus. Another approach yielding greater diversity would be to use multiple alleles
where they exist for each germiine. At this time, there is little or no quantitative data on
allele frequency within the population. When allele frequency becomes available, a more
informed decision can be made regarding the likelihood of tolerance for a specific non-
consensus allele within the target patient population.
[113] When two or more possible substitutions are being evaluated for use at a specific
position when both are found in the human germiine, the decision may become subjective.
In such a case additional information can be incorporated that may reflect different levels of
expression of particular genes (Cox et al. Eur J Immunol. 1994 Apr;24(4):827-36). One
underlying assumption of such a strategy would be that relative expression level of a
particular germiine (or corresponding sequence strings) correlates with the relative
immunogenicity.
[1 14] The sequences used for the method disclosed herein are those of homologous
proteins with sufficient homology to allow their alignment with the protein whose
immunogenicity is being reduced. One might argue that if a particular protein sequence is
found anywhere within the expressed human genome that there is innate tolerance to that
peptide. Such a proposition greatly increases the number of possible sequences that could
be used to reduce the immunogenicity of a protein. In such a case however, alignment of
proteins that are not structurally homologous would likely be prohibitive. In addition, the
processing of a protein to produce the strings to which tolerance is developed may be
structurally determined. Therefore, a specific strings may be nonimmunogenic in its native
context but immunogenic in an altered structural context.
Scoring Functions - String Content
[115] In order to evaluate the fitness of protein variants, amino acid modifications in the
parent protein may be scored using a variety of scoring functions. Central to preferred
embodiment of immunogenicity reduction method described herein is that at least one
scoring function is aimed at maximizing the content of host linear sequence strings that are
present in a set of host sequences. Typically, but not always, a computer is used to score
potential amino acid substitutions.
[116] In one embodiment, substitutions may be scored according to their occupancy in the
set of host sequences, i.e., whether or not a given amino acid is part of the diversity at a
given position. The use of position-specific alignment information to generate a list of
considered amino acids at a variable position is well known in the art; see for example
Lehmann & Wyss, 2001 , Curr Opin Biotechnol 12(4): 371-5; Lehmann et a/., 2000, Biochim
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BiophysActa 1543(2):408-415; Rath & Davidson, 2000, Protein Sc/, 9(1 2):2457-69;
Lehmann ef a/., 2000, Protein Eng 13(1):49-57; Desjarlais & Berg, 1993, Proc Natl Acad Sci
USA 90(6):2256-60; Desjarlais & Berg, 1992, Proteins 12(2):101-4; Henikoff & Henikoff,
2000, Adv Protein Chem 54:73-97; Henikoff & Henikoff, 1 994, J Mol Biol 243(4);574-8.
Thus, for example, for the parent nonhuman VUc sequence aligned to the human sequences
in Figure 1b, substitutions to be considered at position 1 would be Ala, Asp, Glu, Asn, and
Val. In a more preferred embodiment, substitutions are scored based on their frequency in
the set of human sequences listed. For example, in the previous example, Asp and Glu
occur most frequently at position 1, and thus may be more preferable substitutions that Ala,
Asn, or Val. The basis for this scoring function is that the frequency of a given amino acid at
a given position in the alignment is proportional to its potential for being in a host string.
[117] Occupancy and frequency provide relatively straightforward approximations for
designing substitutions that have the potential for reduced immunogenicity. Their use,
however, does not take into account the context of the parent sequence. Although
frequency is proportional to the potential for a substitution to increase host content of a
string, it is not a direct measure. In order to more accurately incorporate the information
present in an aligned set of host sequences into a measure of immunogenicity, an approach
can be taken wherein the linearity or contiguity of a given position in the context of the
strings that comprise it is considered. In this most preferred embodiment, substitutions in a
parent sequence are scored based on the probability of removing a nonhost string and
replacing it with a less immunogenic string, namely one present in the set of host
sequences. This method of scoring may employ the calculation of identity or percent identity
of a parent string to a host string within a window of equivalent positions. In one
embodiment, the identity of a string in sequence s to a host string in sequence h, (IDstring),
can be presented as the sum of amino acid sequence identities in a given window size,
according to equation 1:
Equation 1 IDstring (i 9 w) = atjh
where w is the string window size, i is the first position in the string, aa s j is the amino acid at
position j of sequence s, aa h j is the amino acid at position j of the host sequence h, and the
Kronecker delta function is used to return a value of 1 for a match (for example if the parent
and host amino acids at position j are both serine) and 0 if there is no match (for example if .
the parent amino acid at position j is a serine but the host amino acid is a leucine). Figure
2a illustrates equation 1 using a region of the VH of murine anti-Her2 antibody m4D5
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(VH_m4D5) as the parent sequence s and the homologous region from the VH human
germline (VHM-2) as human sequence h.
[118] In a further embodiment, it is assumed that the most immunologically appropriate
measure of host string content at position i is the maximal identity between a string of
sequence s and any host sequence in the alignment, as calculated in equation 2:
Equation 2 /Z)max = max(Y 8 . >)
where HS is the set of host sequences. In other words, if IDstring at position i is equal to w
for any one of the host sequences, IDmax = w as well, and the i th string is assumed to be
minimally immunogenic. The concept of the IDmax quantity represented by Equation 2 is
illustrated in Figure 2b.
[119] Finally, these equations can be combined to calculate a single numerical metric for
total host string content (HSC) of a sequence s by summing the IDmax values over all
pertinent sequence positions, as in equation 3:
Equation 3 HSC{s) = 100 Y max( Y 8 , h )
where L is the length of the sequence and HS is the set of host sequences in the alignment.
A perfectly host sequence would have an HSC of 100. One might alternatively say that such
a sequence is 100% host. The concept of the HSC quantity represented by Equation 3 is
illustrated in Figure 2c. In alternative embodiments, Equation 3 can be modified further such
that the final score is dependent on the relative usage of each host sequence in the
alignment. Strings from sequences that are more frequently expressed by hosts are
expected to be more tolerized, and therefore may be given correspondingly higher influence
in a scoring system.
[120] In an alternative embodiment, one can measure the exact string content (ESC) as in
Equation 3a:
Equation 3a ESC(s) = 100 : Y max 8 . * ,
(Z-w+1) j~{ h z H s ^^^^i
where the notation aa 5 LI+w _i refers the contiguous sequence string in protein s from position i
to position i+w-1. In this embodiment, only perfect matches of size w are counted in the
score.
[121] It is worth noting that, since the scoring systems in Equations 3 and 3a are based on
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local sequence identity and/or similarity evaluated over windows of defined size, a sequence
with high HSC can be constructed of sequence segments that are maximally similar to
different members of the set of host sequences at different positions.
[122] The above measure of hostness is likely to be more immunologically relevant than
the more commonly used global identity measure of equation 4:
1 1
Equation 4 globallD = 100 -max— Y 8 , b
Equation 4 disregards the extent of contiguous sequence identity, which is particularly
relevant for capturing the molecular behavior of the immune system.
[123] Additional scoring functions similar to equations 3 are also possible. For example, as
will be appreciated by those skilled in the art, there is some uncertainty regarding the
hostness of a string wherein IDmax = w-1, w-2, etc. In one alternative embodiment,
sequence similarity is compared instead of identity, using any of a variety of amino acid
substitution matrices (e.g. PAM, BLOSUM62, etc.), providing a host string similarity (HSS)
score as in equation 5:
Equations HSS(s) = 100 Y maxfe** J J )
L-W+l j% heHS J
where S is a substitution score comparing any two amino acids. In yet another alternative,
sequence identities are weighted according to the extent of identity, as in equation 6:
Equation 6 HSC(s) = 1 00 ■ — - Y /(max( Y S , , ))
(L-w+l) tt °°>' aa >
where f is a continuous or noncontiguous function dependent on IDmax. For example,
perfect matches can be weighted greater than near perfect matches {e.g. f(w)=1 , f(w-1 )=.5,
etc.), and poor matches can be discarded (e.g. f(w-3)=f(w-2) = 0).
String window size
[124J The fundamental binding units of class I and class II MHC proteins are both 9 amino
acids. In a preferred embodiment, the window size w used to create and score parent
sequences is 9. However, it is also known that additional peptide flanking residues (PFRs)
can influence T-cell recognition (via the TCR) of class II MHC-peptide complexes (see for
example Arnold et al., 2002, J Immunology 169(2): 739-49), with the residues at positions P-
1 (one position before the 1 st MHC binding position) and P1 1 being most influential.
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Because these effects might influence immune tolerance, a desirable goal of the invention,
larger window sizes (e.g. 1 2) can be used. It should be noted however, that sequences
optimized with similar window sizes are highly correlated.
Optimization of HSC
[125] Although a definition of string scoring systems is useful, an efficient process for
discovering sequences with high HSC is also desirable. It is therefore a further aspect of the
invention to provide methods for dynamic optimization of HSC given the described scoring
systems.
[1 26] Desirable features of an optimization method include but are not limited to the
following: 1) the output sequences are optimal or near-optimal (subject to design constraints)
in their host string content; 2) structural constraints can be used to modulate the nature of
the optimized sequences; and 3) multiple near-optimal solutions can be generated.
Additionally, in some preferred embodiments, host string content may be maximized using a
minimal number of substitutions.
[1 27] In a preferred embodiment, an iterative algorithm for optimization of HSC works as
follows. 1) a parent sequence and set of host sequences are defined; 2) mutational
constraints are defined at functionally or structurally important positions, referred to herein as
masking - in a preferred embodiment, for antibody applications, positions within or
structurally proximal to CDR residue (as defined by herein, or alternatively as defined by
Kabat or Chothia) and/or interface are masked, locked, or fixed so that mutations are not
possible (in some embodiments this constraint can be relaxed if the potential mutation is a
conservative substitution of the parent amino acid). In a preferred embodiment, positions
within 5 angstroms of a CDR residue or interface are masked. In other preferred
embodiments, positions within 6.5 angstroms of a CDR residue or interface are masked; 3)
host sequence segments (up to a defined length: lengths from 1-6 are typical) are collected
from the alignment and stored for each position: segments that violate the mutational
constraints are not collected; 4) each segment is analyzed for its potential impact on HSC, in
the context of the current parent sequence, defined as String Impact (SI) in equation 7:
Equation 7 & ~+ < z » = HSC ^ )) ~ HSC{parenf) ^
where y m (z) is a host segment of length z replacing segment x at position m, and s(y m ) and
. parent are versions of the parent sequence that include these segments (the parent
sequence contains xjz)). 5) a single string is randomly selected from all stored host strings;
the probability of selection is biased and proportional to the impact on HSC and inversely
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proportional to the number of mutations relative to the current parent sequence, as in
Equation 8:
Paz sr(*M-+yM)
Equation 8 ****
This selected string is substituted into the current parent sequence for its corresponding
parent amino acids on an amino acid string by amino acid string basis. This kind of selection
bias tends to optimize host string content with minimal perturbation of the original sequence.
6) steps 4 and 5 are repeated until no further optimization is possible (no segment
substitutions have a favorable impact on host string content).
[128] Such an algorithm is inherently non-deterministic, so independent runs of the
algorithm will tend to generate different solutions (this is a favorable feature). In a preferred
embodiment, such an algorithm is applied numerous times to generate a diverse array of
unique solutions. These solutions can be further clustered such that representative
sequences can be prioritized for further analysis. For example, in one embodiment, the
solution sequences are clustered into groups of similar sequences according to mutational
distance, using a nearest neighbor single linkage hierarchical clustering algorithm to assign
sequences to related groups based on similarity scores. Clustering algorithms may be
useful for classifying sequences into representative groups. Representative groups may be
defined, for example, by similarity. Measures of similarity include, but are not limited to
sequence similarity and energetic similarity. Thus the output sequences from computational
screening may be clustered around local minima, referred to herein as clustered sets of
sequences. Sets of sequences that are close in sequence space may be distinguished from
other sets. In one embodiment, diversity across clustered sets of sequences may be
sampled by experimentally testing only a subset of sequences within each clustered set. For.
example, all or most of the clustered sets could be broadly sampled by including the lowest
energy sequence from each clustered set of sequences to be experimentally tested.
Because the sequence space of solutions with optimized HSC can be large, additional
methods can be applied to ensure that a broad set of sequences is created. In a preferred
embodiment, individual framework sequences generated by the procedure are clustered
separately to generate a list of nonredundant basis framework regions (FRs) with high HSC.
These basis FRs are then computationally assembled in all combinations along with the
CDRs to generate a secondary list of solution sequences (which will usually have some
overlap with the primary set). Alternatively, the basis FRs may be combined into an
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experimental library, for example a combinatorial library.
Framework Diversity
[129] Application of this algorithm will generate variant protein solutions for which HSC is
higher than the original parent sequence. It will also frequently generated solutions in which
substituted strings are derived from different members of the alignment. The variant
sequences derived using the present invention generally have unique properties relative to
sequences generated using other methodologies. For example, in the context of an
antibody, the protein variants of the invention frequently derive their host string content from
a combination of different host germline sequences. This may be true even within a single
FR. Quantification of these properties is useful for defining the nature of sequences derived
using the present invention. A clear distinction emerges from a comparison of exact string
content (meaning a perfect match over window w) in any single germline sequence versus
exact w-mer string content within the set of all germline sequences (content of strings for
which IDmax = w). Single germline exact string content (SGESC) of a variant sequence v
may thus be defined as:
Equation 9 SGESCCv) = 100 max Y 8
M
[130] This quantity provides the extent to which a string-optimized sequence has string
identity with the closest single germline sequence. Using this definition, it is also possible to
assess the extent to which the high host string content of a given variant sequence v is
derived from a single germline as opposed to multiple germline sequences. Framework
region homogeneity (FRH) is defined as follows:
Equation 1 0 FRH(v) = - , ^ ,
w ESC(y) 1 ^
x max Y 5 „ h
_ SGESC(y) _ heHs ^ °°L^,™L^x
Smax£ , „
[131] In other words, if a variant sequence's exact string content is derived solely from a
single germline sequence, the FRH would be close to 1.0. It should be noted that a similar
or identical quantity can be defined for non-antibody proteins. Alternatively, as is the case
with many of the variant sequences created by the present invention, FRH values can be
significantly less than 1, with values ranging from 0.4 to 1.0, indicating, as expected, that
sequences with high exact string content can be discovered with contributions from multiple
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germline subfamilies and sequences. As described more fully in Example 5 below, variant
sequences generated using the present invention have high HSC values yet many have low
FRH values, indicating their HSC is derived from multiple germline frameworks.
Additional Scoring
[132] The above methods of scoring use the information present in an aligned set of host
sequences as a metric of irnmunogenicity to maximize the content of host linear sequence
strings in a parent sequence. In addition to such scoring functions, other scoring functions
and methods may be employed. Such additional scoring functions may be aimed at the
same goal as the aforementioned linear string scoring function, namely irnmunogenicity
reduction of the parent protein. Alternatively, such additional scoring functions may be used
to achieve other goals, for example optimization of protein stability, solubility, expression,
pharmacokinetics, and/or aspects of protein function such as affinity of the parent protein for
a target ligand, specificity, effector function, and/or enzymatic activity. For example an
additional scoring function may be employed to enhance the affinity of an antibody variable
domain for its target antigen. Such additional scoring functions may be employed statically
or dynamically for the generation of optimized protein variants. A number of embodiments
are described below as preferred additional scoring functions that may be used with the
aforementioned linear string scoring method of the present invention. However, these are
not meant to constrain the invention to these embodiments, and it should be clear that any
method of scoring the fitness of an amino acid modification in a parent protein may be
coupled with the novel linear string scoring method of the present invention so that optimal
protein variants may be designed.
[133] In a preferred embodiment, substitutions are scored based on their structural
compatibility with the structure of the parent protein. Such methods of scoring may require
the structural coordinates that describe the three-dimensional structure of the protein, for
example as obtained by X-ray crystallographic and nuclear magnetic resonance (NMR)
techniques. Suitable proteins structures may also be obtained from structural models, which
may be generated by methods that are known in the art of structural biology, including but
not limited to de novo and homology modeling. Structure-based scoring functions may
include any number of potentials that describe or approximate physical or chemical energy
terms, including but not limited to a van der Waals potential, a hydrogen bond potential, an
atomic solvation potential or other solvation models, a secondary structure propensity
potential, an electrostatic potential, a torsional potential, an entropy potential, and/or
additional energy terms. In other preferred embodiments, scoring methods may also be
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derived from sequence information, including but not limited to knowledge-based potentials
derived from protein sequence and/or structure statistics, threading potentials, reference
energies, pseudo energies, homology-based energies, and sequence biases derived from
sequence alignments. In alternately preferred embodiments, both structural and sequence-
based potentials are used to generate one or more scoring functions that may be coupled
with the linear string scoring method of the present invention.
[134] In a most preferred embodiment, a scoring method is used wherein the structural and
functional integrity of substitutions are evaluated using a sequence and structure-based
scoring function described in USSN 60/528,229, filed December 8, 2003, entitled Protein
Engineering with Analogous Contact Environments; and USSN 60/602,566, filed August 17,
2004, entitled Protein Engineering with Analogous Contact Environments. This method
combines sequence alignment information and structural information to predict the structural
compatibility of one or more substitutions with a protein structure template. Nearest
neighbor structure-based scores generated by this method include Structural Consensus
and Structural Precedence as provided in the Examples. This method is particularly well
suited for application to evaluating the structural fitness of immunoglobulins due to their
substantial sequence and structural homology.
[135] In a preferred embodiment, substitutions are scored using a scoring function or
computational design program that is substantially similar to Protein Design Automation®
(PDA®) technology, as is described in US 6,188,965; US 6,269,312; US 6,403,312; US
6,708,120; US 6,804,61 1; US 6,792,356; USSN 09/782,004; USSN 09/812,034; USSN
09/927,790; USSN 10/218,102; USSN 10/101,499; USSN 10/218,102; USSN 10/666,311;
USSN 10/665,307; USSN 10/888,748; PCT WO 98/07254; PCT WO 99/24229; PCT WO
01/40091; and PCT WO 02/25588. In another preferred embodiment, a computational
design method substantially similar to Sequence Prediction Algorithm™ (SPA™) technology
is used, as is described in (Raha et a/., 2000, Protein ScL 9: 1106-1119), USSN 09/877,695,
and USSN 10/071,859. In another preferred embodiment, the computational methods
described in USSN 10/339,788, are used.
[136] In another preferred embodiment, optimized sequences are also assessed for
surface similarity with host antibodies. Ensuring similarity may be important for reducing the
probability of introducing novel 3D epitopes, which are potentially recognized by B-cell
receptors. In a preferred embodiment, surface similarity at position i is quantified as follows:
Equation 11 surfscoreii) = maxe Jm}
keHS
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where fj exp is the fraction accessibility of position i to solvent, proximity(i j) is the spatial
proximity of positions i and j in the three-dimensional structure of the protein, S is a measure
of amino acid similarity, and T is a temperature factor used to tune the stringency of the
similarity comparison. It will be appreciated from the equation that if sequences are identical
in the region of position i, a surfscore of 1.0 will be approached. Alternatively, a score of 1.0
can also be achieved if a position is completely buried (i.e. fi exp =0), since the position would
not be accessible to B-cell receptors. Lower scores represent surface positions for which
there are significant differences between the variant sequence and the most similar host
sequence. In a preferred embodiment, the proximity between two positions is inversely
related to their distance (e.g. a Gaussian or exponential function of the distance), and the
proximity of a position to itself is 1 .0. In a preferred embodiment, the decay of the proximity
function is tuned such that patches of positions correspond to the size of a typical antibody
epitope. ,
[137] Other surface properties may also be desirable. For example, optimized variant
sequences may be assessed for the exposure of nonpolar amino acids, which is generally
expected to decrease solubility. In such cases, variant sequences with lower nonpolar
exposure can be prioritized over alternatives. Surface electrostatic properties may also be
assessed for variant sequences. In a preferred embodiment, surface properties are
assessed for multiple variants with optimized HSC, in order to limit the set of variants that
will be experimentally screened.
[138] In another embodiment, substitution matrices or other knowledge-based scoring
methods are used to identify alternate sequences that are likely to retain the structure and
function of the protein. Such scoring methods can be used to quantify how conservative a
given substitution or set of substitutions is. In most cases, conservative mutations do not
significantly disrupt the structure and function of proteins (see for example, Bowie et a/.,
1990, Science 247: 1306-1310, Bowie & Sauer, 1989, Proc. Nat Acad, ScL USA 86: 2152-
2156, and Reidhaar-Olson & Sauer, 1990, Proteins 7: 306-316). However, non-conservative
mutations can destabilize protein structure and reduce activity (see for example, Lim et a/.,
1992, Biochem. 31: 4324-4333). Substitution matrices including but riot limited to
BLOSUM62 provide a quantitative measure of the compatibility between a sequence and a
target structure, which can be used to predict non-disruptive substitution mutations (Topham
et a/., 1997, Prot Eng. 10: 7-21). The use of substitution matrices to design peptides with
improved properties has been disclosed (Adenot etal., 1999, J. Mol. Graph. Model. 17: 292-
309). Substitution matrices include, but are not limited to, the BLOSUM matrices (Henikoff &
Henikoff, 1992, Proc. Nat. Acad. Sci. USA 89: 10917, the PAM matrices, the Dayhoff matrix,
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and the like. For a review of substitution matrices, see for example Henikoff, 1 996, Curr.
Opin. Struct Biol. 6: 353-360. It is also possible to construct a substitution matrix based on
an alignment of a given protein of interest and its homologs; see for example Henikoff &
Henikoff, 1996, Comput. Appl. BioscL 12: 135-143.
[139] In a preferred embodiment, other methods for scoring immunogenicity may
additionally be used. Most preferably, immunogenicity may be scored using a function that
considers peptide binding to one or more MHC molecules. For example, substitutions would
be scored such that there are no or a minimal number of immune epitopes that are predicted
to bind, with high affinity, to any prevalent MHC alleles. These methods of scoring may be
useful, for example, for designing substitutions in VH CDR3, for which scoring using human
germline strings may be less straightforward. Several methods of identifying MHC-binding
epitopes in protein sequences are known in the art and may be used to score epitopes in an
antibody. See for example WO 98/52976; WO 02/079232; WO 00/3317; USSN 09/903,378;
USSN 10/039,170; USSN 60/222,697; USSN 10/339788; PCTWO 01/21823; and PCT WO
02/00165; Mallios, 1999, Bioinformatics 15: 432-439; Mallios, 2001, Bioinformatics 17: 942-
948; Stumiolo et a/., 1999, Nature Biotech. 17: 555-561; WO 98/59244; WO 02/069232; WO
02/77187; Marshall etal., 1995, J. Immunol 154: 5927-5933; and Hammer et aA, 1994, J.
Exp. Med. 180: 2353-2358. Sequence-based information can be used to determine a
binding score for a given peptide - MHC interaction (see for example Mallios, 1999,
Bioinformatics 15: 432-439; Mallios, 2001 , Bioinformatics 17: p942-948; Sturniolo et. a/.,
1999, Nature Biotech. 17: 555-561). It is possible to use structure-based methods in which a
given peptide is computationally placed in the peptide-binding groove of a given MHC
molecule and the interaction energy is determined (for example, see WO 98/59244 and WO
02/069232). Such methods may be referred to as "threading" methods. Alternatively, purely
experimental methods can be used; for example a set of overlapping peptides derived from
the protein of interest can be experimentally tested for the ability to induce T-cell activation
and/or other aspects of an immune response, (see for example WO 02/77187). In a
preferred embodiment, MHC-binding propensity scores are calculated for each 9-residue
frame along the protein sequence using a matrix method (see Sturniolo et at, supra]
Marshall et a/., 1995, J. Immunol. 154: 5927-5933, and Hammer et a/., 1994, J. Exp. Med.
180: 2353-2358). It is also possible to consider scores for only a subset of these residues,
or to consider also the identities of the peptide residues before and after the 9-residue frame
of interest. The matrix comprises binding scores for specific amino acids interacting with the
peptide binding pockets in different human class II MHC molecule. In the most preferred
embodiment, the scores in the matrix are obtained from experimental peptide binding
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studies. In an alternate preferred embodiment, scores for a given amino acid binding to a
given pocket are extrapolated from experimentally characterized alleles to additional alleles
with identical or similar residues lining that pocket. Matrices that are produced by
extrapolation are referred to as "virtual matrices".
[140] In alternate embodiments, additional scoring functions are employed that predict
reactive sites within a protein, such as deamidation sites, glycosylation sites, oxidation sites,
protealytic cleavage sites, and the like.
[141] It will be appreciated by one of skill in the art that the use of combinations of any of
the aforementioned scoring functions and/or other scoring functions is contemplated. In one
embodiment, this could be accomplished by evaluating the outputs of the results from
separate calculations. Alternatively, scoring functions may be combined into one scoring
term. This latter strategy enables different scoring terms to be weighted separately, thus
providing more control over the relative contributions of the scoring terms, and a greater
capacity to tune the scoring function for a desired engineering strategy.
Additional Optimization of Sequences
[142] In a preferred embodiment, after optimized protein variants have been engineered
using the aforementioned scoring functions, additional optimization of protein variants may
be carried out. In this way, an optimized protein variant can be thought of as primary variant
or template for further optimization, and variants of this primary variant can be thought of as
secondary variants. Because variant sequences of the invention are preferably derived from
a HSC-increasing procedure in which substitution of structurally important positions is
disallowed or discouraged (for example masking), it is likely that additional optimization of
HSC is possible if those positions are allowed to vary in a secondary analysis. Optimization
of other properties is possible, including but are not limited to protein affinity, expression,
specificity, solubility, activity, and effector function. Thus the variant sequences derived in
the primary analysis can represent variants for further optimization. In a preferred
embodiment, the secondary analysis comprises the steps of 1) string analysis of the
template sequence to identify secondary amino acid diversity that will have neutral, positive,
or minimal impact on HSC; 2) experimental production of secondary variant sequences
using the diversity derived in step 1 ; and 3) experimental screening of secondary variant
sequences.
[143] For these purposes, the string impact (SI) of a single substitution at position m from
amino acid x to amino acid y can be quantified as in Equation 7 (where segment length z =
1). As will be appreciated, the maximum possible string increase (for a single substitution at
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an internal position) is w and the maximum possible decrease is w.
[144] In a most preferred embodiment, substitutions in a primary variant are chosen as
those substitutions that will result in zero or positive string impact, referred to herein as string
neutral and string positive substitutions respectively. In other embodiments, substitutions
that result in negative string impact, i.e. string negative substitutions, may also be
considered for engineering secondary variants. Secondary variants then may be
constructed, expressed, and tested experimentally. Secondary substitutions that show
favorable properties with respect to antigen affinity, effector function, stability, solubility,
expression, and the like, may be combined in subsequent variants to generate a more
optimized therapeutic candidate.
Optimization of Non-Xenogeneic Proteins
[145] It can be appreciated that the optimization described above is not restricted to
immunogenicity reduction of xenogeneic proteins. Evaluation of potential substitutions for
string impact provides an excellent strategy for generating substitution diversity for
engineering protein variants with optimized properties. A clear advantage of this approach is
that it generates protein variants with minimal immunogenicity risk, the importance of which
has been discussed extensively and is a primary goal of the present invention. An additional
advantage of this approach is that because the sequences being used to evaluate string
impact are typically derived from a set of naturally evolved host sequences, variants
designed are effectively enriched for stability, solubility, and other favorable properties. The
utility of this capability lies in the fact that there are innumerable amino acid modifications
that are detrimental or deleterious to proteins. By screening a quality set of variant diversity,
the chances are increased that a protein variant of the desired property will be obtained.
The capacity of the string impact approach to generate a quality set of variant diversity
derives from the greater tolerance to mutation of positions which sample greater diversity,
and the greater propensity of amino acids in a set of naturally evolved sequences to be
compatible with a homologous protein's structure, stability, solubility, function, and the like.
[146] This string impact approach to variant design may be applied not only to the
generation of secondary variants as described above, but may also be used to engineer
amino acid modifications in proteins that are presumably already minimally immunogenic.
This may include, for example, natural host proteins. Alternatively, and in a preferred
embodiment, the string impact strategy may be applied to engineer modifications in an
antibody variable region (VH or VL) that is humanized (Clark, 2000, Immunol Today 21 :397-
402); or "fully human" as obtained for example using transgenic mice (Bruggemann et a/.,
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1997, Curr Opin Biotechnol 8:455-458) or human antibody libraries coupled with selection
methods (Griffiths et a/., 1998, Curr Opin Biotechnol 9:102-108). As with optimization of
primary variant sequences described above, the string impact analysis described here can
be used to identify secondary diversity that will have neutral, positive, or minimal impact on
HSC, as well as potentially other favorable properties. Such diversity can then be used to
screen for optimized versions of these sequences without increasing the risk of
immunogenicity.
Experimental Production, Screening, and Testing
[147] Methods for production and screening of protein variants are well known in the art.
General methods for antibody molecular biology, expression, purification, and screening are
described in Antibody Engineering, edited by Duebel & Kontermann, Springer-Verlag,
Heidelberg, 2001 ; and Hayhurst & Georgiou, 2001 , Curr Opin Chem Biol 5:683-689;
Maynard & Georgiou, 2000, Annu Rev Biomed Eng 2:339-76. Also see the methods
described in USSN 10/339788, filed on March 3, 20O3, USSN 10/672,280, filed September
29, 2003, and USSN 1 0/822,231 , filed March 26, 2004.
[148] |n one embodiment of the present invention, the library sequences are used to create
nucleic acids that encode the member sequences, and that may then be cloned into host
cells, expressed and assayed, if desired. These practices are carried out using well-known
procedures, and a variety of methods that may find use in the present invention are
described in Molecular Cloning - A Laboratory Manual, 3 rd Ed. (Maniatis, Cold Spring Harbor
Laboratory Press, New York, 2001), and Current Protocols in Molecular Biology (John Wiley
& Sons). The nucleic acids that encode the protein variants of the present invention may be
incorporated into an expression vector in order to express the protein. Expression vectors
typically comprise a protein operably linked, that is placed in a functional relationship, with
control or regulatory sequences, selectable markers, any fusion partners, and/or additional
elements. The protein variants of the present invention may be produced by culturing a host
cell transformed with nucleic acid, preferably an expression vector, containing nucleic acid
encoding the protein variants, under the appropriate conditions to induce or cause
expression of the protein. A wide variety of appropriate host cells may be used, including
but not limited to mammalian cells, bacteria, insect cells, and yeast. For example, a variety
of cell lines that may find use in the present invention are described in the ATCC cell line
catalog, available from the American Type Culture Collection. The methods of introducing
exogenous nucleic acid into host cells are well known in the art, and will vary with the host
cell used.
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[149] In a preferred embodiment, protein variants are purified or isolated after expression.
Proteins may be isolated or purified in a variety of ways known to those skilled in the art.
Standard purification methods include chromatographic techniques, electrophoretic,
immunological, precipitation, dialysis, filtration, concentration, and chromatofocusing
techniques. As is well known in the art, a variety of natural proteins bind antibodies, for
example bacterial proteins A, G, and L, and these proteins may find use in the present
invention for purification. Purification can often be enabled by a particular fusion partner.
For example, proteins may be purified using glutathione resin if a GST fusion is employed,
Nf 2 affinity chromatography if a His-tag is employed, or immobilized anti-flag antibody if a
flag-tag is used. For general guidance in suitable purification techniques, see Protein
Purification: Principles and Practice, 3 rd Ed., Scopes, Springer-Verlag, NY, 1994.
[150] Protein variants may be screened using a variety of methods, including but not
limited to those that use in vitro assays, in vivo and cell-based assays, and selection
technologies. Automation and high-throughput screening technologies may be utilized in the
screening procedures. Screening may employ the use of a fusion partner or label, for
example an immune label, isotopic label, or small molecule label such as a fluorescent or
colorimetric dye.
[151] In a preferred embodiment, the functional and/or biophysical properties of protein
variants are screened in an in vitro assay. In a preferred embodiment, the protein is
screened for functionality, for example its ability to catalyze a reaction or its binding affinity to
its target. Binding assays can be carried out using a variety of methods known in the art,
including but not limited to FRET (Fluorescence Resonance Energy Transfer) and BRET
(Bioluminescence Resonance Energy Transfer) -based assays, AlphaScreen™ (Amplified
Luminescent Proximity Homogeneous Assay), Scintillation Proximity Assay, ELISA
(Enzyme-Linked Immunosorbent Assay), SPR (Surface Plasmoh Resonance, also known as
BIACORE®), isothermal titration calorimetry, differential scanning calorimetry, gel
electrophoresis, and chromatography including gel filtration. These and other methods may
take advantage of some fusion partner or label. Assays may employ a variety of detection
methods including but not limited to chromogenic, fluorescent, luminescent, or isotopic
labels. The biophysical properties of proteins, for example stability and solubility, may be
screened using a variety of methods known in the art. Protein stability may be determined
by measuring the thermodynamic equilibrium between folded and unfolded states. For
example, protein variants of the present invention may be unfolded using chemical
denaturant, heat, or pH, and this transition may be monitored using methods including but
not limited to circular dichroism spectroscopy, fluorescence spectroscopy, absorbance
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spectroscopy, NMR spectroscopy, calorimetry, and proteolysis. As will be appreciated by
those skilled in the art, the kinetic parameters of the folding and unfolding transitions may
also be monitored using these and other techniques. The solubility and overall structural
integrity of a protein variant may be quantitatively or qualitatively determined using a wide
range of methods that are known in the art. Methods which may find use in the present
invention for characterizing the biophysical properties of protein variants include gel
electrophoresis, chromatography such as size exclusion chromatography and reversed-
phase high performance liquid chromatography, mass spectrometry, ultraviolet absorbance
spectroscopy, fluorescence spectroscopy, circular dichroism spectroscopy, isothermal
titration calorimetry, differential scanning calorimetry, analytical ultra-centrifugation, dynamic
light scattering, proteolysis, and cross-linking, turbidity measurement, filter retardation
assays, immunological assays, fluorescent dye binding assays, protein-staining assays,
microscopy, and detection of aggregates via ELISA or other binding assay. Structural
analysis employing X-ray crystallographic techniques and NMR spectroscopy may also find
use.
1152) In a preferred embodiment, protein variants are screened using one or more cell-
based or in vivo assays. For such assays, purified or unpurified proteins are typically added
exogenously such that cells are exposed to individual variants or pools of variants belonging
to a library. These assays are typically, but not always, based on the function of the protein;
that is, the ability of the protein to bind to its target and mediate some biochemical event, for
example effector function, ligand/receptor binding inhibition, apoptosis, and the like. Such
assays often involve monitoring the response of cells to the protein, for example cell survival,
cell death, change in cellular morphology, or transcriptional activation such as cellular
expression of a natural gene or reporter gene. For example, such assays may measure the
ability of antibody variants to elicit ADCC, ADCP, or CDC. For some assays additional cells
or components, that is in addition to the target cells, may need to be added , for example
serum complement, or effector cells such as peripheral blood monocytes (PBMCs), NK cells,
macrophages, and the like. Such additional cells may be from any organism, preferably
humans, mice, rat, rabbit, and monkey. Proteins may cause apoptosis of certain cell lines
expressing the target, or they may mediate attack on target cells by immune cells which
have been added to the assay. Methods for monitoring cell death or viability are known in
the art, and include the use of dyes, immunochemical, cytochemical, and radioactive
reagents. For example, caspase staining assays may enable apoptosis to be measured,
and uptake or release of radioactive substrates or fluorescent dyes such as alamar blue may
enable cell growth or activation to be monitored. In a preferred embodiment, the DELFIA®
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EuTDA-based cytotoxicity assay (Perkin Elmer, MA) is used. Alternatively, dead or
damaged target cells may be monitored by measuring the release of one or more natural
intracellular proteins, for example lactate dehydrogenase. Transcriptional activation may
also serve as a method for assaying function in cell-based assays. In this case, response
may be monitored by assaying for natural genes or proteins which may be upregulated, for
example the release of certain interleukins may be measured, or alternatively readout may
be via a reporter construct. Cell-based assays may also involve the measure of
morphological changes of cells as a response to the presence of a protein. Cell types for
such assays may be prokaryotic or eukaryotic, and a variety of cell lines that are known in
the art may be employed. Alternatively, cell-based screens are performed using cells that
have been transformed or transfected with nucleic acids encoding the variant proteins. That
is, protein variants are not added exogenously to the cells. For example, in one
embodiment, the cell-based screen utilizes cell surface display. A fusion partner can be
employed that enables display of variants on the surface of cells (Witrrup, 2001 , Curr Opin
> Biotechnol, 12:395-399).
[153] As is known in the art, a subset of screening methods are those that select for
favorable members of a library. The methods are herein referred to as " selection methods ",
and these methods find use in the present invention for screening protein variants. When
protein libraries are screened using a selection method, only those members of a library that
are favorable, that is which meet some selection criteria, are propagated, isolated, and/or
observed. As will be appreciated, because only the most fit variants are observed, such
methods enable the screening of libraries that are larger than those screenable by methods
that assay the fitness of library members individually. Selection is enabled by any method,
technique, or fusion partner that links, covalently or noncovalently, the phenotype of a
protein with its genotype, i.e., the function of a protein with the nucleic acid that encodes it
For example the use of phage display as a selection method is enabled by the fusion of
library members to the gene III protein. In this way, selection or isolation of protein variants
that meet some criteria, for example binding affinity to the protein's target, also selects for or
isolates the nucleic acid that encodes it. Once isolated, the gene or genes encoding variants
may then be amplified. This process of isolation and amplification, referred to as panning,
may be repeated, allowing favorable protein variants in the library to be enriched. Nucleic
acid sequencing of the attached nucleic acid ultimately allows for gene identification.
[154] A variety of selection methods are known in the art that may find use in the present
invention for screening protein libraries. These include but are not limited to phage display
(Phage display of peptides and proteins: a laboratory manual, Kay et ai t 1996, Academic
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Press, San Diego, CA, 1996; Lowman etal., 1991, Biochemistry 30:10832-10838; Smith,
1985, Science 228:1315-1317) and its derivatives such as selective phage infection
(Malmborg et al. y 1997, J Mol Biol 273:544-551), selectively infective phage (Krebber et a/.,
1997, J Mol Biol 268:619-630), and delayed infectivity panning (Benhar et a/., 2000, J Mol
Biol 301 :893-904), cell surface display (Witrrup, 2001, Curr Opin Biotechnol, 12:395-399)
such as display on bacteria (Georgiou et al., 1997, Nat Biotechnol 15:29-34; Georgiou et al.,
1993, Trends Biotechnol 1 1 :6-10; Lee et al., 2000, Nat Biotechnol 18:645-648; Jun ef al.,
1998, Nat Biotechnol 16:576-80), yeast (Boder & Wittrup, 2000, Methods Enzymol 328:430-
44; Boder & Wittrup, 1997, Nat Biotechnol 1 5:553-557), and mammalian cells (Whitehorn ef
al., 1995, BioAechnology 13:1215-1219), as well as in vitro display technologies (Amstutz ef
al., 2001 , Curr Opin Biotechnol 12:400-405) such as polysome display (Mattheakis et al.,
1994, Proc Natl Acad Sci USA 91:9022-9026), ribosome display (Hanes et al., 1997, Proc
Natl Acad Sci USA 94:4937-4942), mRNA display (Roberts & Szostak, 1997, Proc Natl Acad
Sci USA 94:12297-12302; Nemoto etal., 1997, FEBS Lett 414:405-408), and ribosome-
inactivation display system (Zhou etal., 2002, J Am Chem Soc 124, 538-543).
[1 55] Other selection methods that may find use in the present invention include methods
that do not rely on display, such as in vivo methods including but not limited to periplasmic
expression and cytometric screening (Chen ef ai, 2001 , Nat Biotechnol 19:537-542), the
protein fragment complementation assay (Johnsson & Varshavsky, 1994, Proc Natl Acad Sci
USA 91 :10340-10344; Pelletier ef al., 1998, Proc Natl Acad Sci USA 95:12141-12146), and
the yeast two hybrid screen (Fields & Song, 1989, Nature 340:245-246) used in selection
mode (Visintin etal., 1999, Proc Natl Acad Sci USA 96:11723-11728). In an alternate
embodiment, selection is enabled by a fusion partner that binds to a specific sequence on
the expression vector, thus linking covalently or noncovalently the fusion partner and
associated variant library member with the nucleic acid that encodes them. For example,
USSN 09/642,574; USSN 10/080.376; USSN 09/792,630; USSN 10/023,208; USSN
09/792,626; USSN 10/082,671; USSN 09/953,351; USSN 10/097,100; USSN 60/366,658;
PCT WO 00/22906; PCT WO 01/49058; PCT WO 02/04852; PCT WO 02/04853; PCT WO
02/08023; PCT WO 01/28702; and PCT WO 02/07466 describe such a fusion partner and
technique that may find use in the present invention. In an alternative embodiment, in vivo
selection can occur if expression of the protein imparts some growth, reproduction, or
survival advantage to the cell.
[1 56] A subset of selection methods referred to as "directed evolution" methods are those
that include the mating or breading of favorable sequences during selection, sometimes with
the incorporation of new mutations. As will be appreciated by those skilled in the art,
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directed evolution methods can facilitate identification of the most favorable sequences in a
library, and can increase the diversity of sequences that are screened. A variety of directed
evolution methods are known in the art that may find use in the present invention for
screening protein variants, including but not limited to DNA shuffling (PCT WO 00/42561 A3;
PCT WO 01/70947 A3), exon shuffling (US 6,365,377; Kolkman & Stemmer, 2001, Nat
Biotechnol 19:423-428), family shuffling (Crameri et a/., 1998, Nature 391:288-291; US
6,376,246), RACHITT™ (Coco et a/., 2001 , Nat Biotechno\ 19:354- 359; PCT WO 02/06469),
STEP and random priming of in vitro recombination (Zhao ef a/., 1998, Nat Biotechnol
16:258-261; Shao et a/., 1998, Nucleic Acids Res 26:681-683), exonuclease mediated gene
assembly (US 6,352,842; US 6,361,974), Gene Site Saturation Mutagenesis™ (US
6,358,709), Gene Reassembly™ (US 6,358,709), SCRATCHY (Lutz et a!., 2001, Proc Natl
Acad Sci USA 98:1 1248-1 1253), DNA fragmentation methods (Kikuchi et a/., Gene 236:159-
167), single-stranded DNA shuffling (Kikuchi et a/., 2000, Gene 243:133-137), and
AMEsystem™ directed evolution protein engineering technology (Applied Molecular
Evolution) (US 5,824,514; US 5,817,483; US 5,814,476; US 5,763,192; US 5,723,323).
[157] In a preferred embodiment, the immunogenicity of the protein variants is determined
experimentally to confirm that the variants do have reduced or eliminated immunogenicity
relative to the parent protein. Several methods can be used for experimental confirmation of
epitopes. In a preferred embodiment, ex vivo T-cell activation assays are used to
experimentally quantitate immunogenicity. In this method, antigen presenting celts and
naive T cells from matched donors are challenged with a peptide or whole protein of interest
one or more times. Then, T cell activation can be detected using a number of methods, for
example by monitoring production of cytokines or measuring uptake of tritiated thymidine. In
the most preferred embodiment, interferon gamma production is monitored using Elispot
assays (Schmittel et al, 2000, J. Immunol. Meth., 24: 17-24). If sera are available from
patients who have raised an immune response to protein, it is possible to detect mature T
cells that respond to specific epitopes. In a preferred embodiment, interferon gamma or IL-5
production by activated T-cells is monitored using Elispot assays, although it is also possible
to use other indicators of T cell activation or proliferation such as tritiated thymidine
incorporation or production of other cytokines. Other suitable T cell assays include those
disclosed in Meidenbauer ef a/., 2000, Prostate 43, 88-100; Schultes & Whiteside, 20O3, J.
Immunol. Methods 279, 1-15; and Stickler et al., 200, J. Immunotherapy, 23, 654-660. In a
preferred embodiment, the PBMC donors used for the above-described T cell activation
assays will comprise class II MHC alleles that are common in patients requiring treatment for
protein responsive disorders. For example, for most diseases and disorders, it is desirable
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to test donors comprising all of the alleles that are prevalent in the population. However/for
diseases or disorders that are linked with specific MHC alleles, it may be more appropriate to
focus screening on alleles that confer susceptibility to protein responsive disorders. In a
preferred embodiment, the MHC haplotype of PBMC donors or patients that raise an
immune response to the wild type or protein variant are compared with the MHC haplotype
of patients who do not raise a response. This data may be used to guide preclinical and
clinical studies as well as aiding in identification of patients who will be especially likely to
respond favorably or unfavorably to the protein therapeutic.
[1 58] In an alternate preferred embodiment, immunogenicity is measured in transgenic
mouse systems. For example, mice expressing fully or partially human class II MHC
molecules may be used. In an alternate embodiment, immunogenicity is tested by
administering the protein variants to one or more animals, including rodents and primates,
and monitoring for antibody formation. Nonhuman primates with defined MHC haplotypes
may be especially useful, as the sequences and hence peptide binding specificities of the
MHC molecules in nonhuman primates may be very similar to the sequences and peptide
binding specificities of humans. Similarly, genetically engineered mouse models expressing
human MHC peptide-binding domains may be used (see for example Sonderstrup et al.,
1999, Immunol. Rev. 172: 335-343; and Forsthuber era/., 2001, J. Immunol. 167: 119-125).
[1 59] The biological properties of the proteins of the present invention may be
characterized in cell, tissue, and whole organism experiments. As is known in the art, drugs
are often tested in animals, including but not limited to mice, rats, rabbits, dogs, cats, pigs,
and monkeys, in order to measure a drug's efficacy for treatment against a disease or
disease model, or to measure a drug's pharmacokinetics, toxicity, and other properties. The
animals may be referred to as disease models. Therapeutics are often tested in mice,
including but not limited to nude mice, SCID mice, xenograft mice, and transgenic mice
(including knockins and knockouts). Such experimentation may provide meaningful data for
determination of the potential of the protein to be used as a therapeutic. Any organism,
preferably mammals, may be used for testing. For example because of their genetic
similarity to humans, monkeys can be suitable therapeutic models, and thus may be used to
test the efficacy, toxicity, pharmacokinetics, or other property of the proteins of the present
invention. Tests of the in humans are ultimately required for approval as drugs, and thus of
course these experiments are contemplated. Thus the proteins of the present invention may
be tested in humans to determine their therapeutic efficacy, toxicity, immunogenicity,
pharmacokinetics, and/or other clinical properties.
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[160] In one embodiment of the present invention, a variant antibody for a host as
compared to a parent antibody includes two or more amino acid substitutions derived from
two or more natural antibodies. In this embodiment, a first resultant variant string in the
variant antibody is rendered most homologous to a first natural antibody, a second resultant
variant string in the variant antibody is rendered most homologous to the corresponding
string in an second natural antibody, the substitutions are not in a CDR, and at least one
resultant string is not a consensus of homologous natural sequences. In a preferred
embodiment, the variant strings in the variant antibody do not include CDR residues. In a
further preferred embodiment, the first and second natural antibodies are from different
subfamilies.
[161] In another embodiment of the present invention, a variant antibody for a host as
compared to a parent antibody includes two or more amino acid substitutions derived from
three or more natural antibodies. In this embodiment, a first resultant variant string in the
variant antibody is rendered most homologous to a first natural antibody, a second resultant
variant string in the variant antibody is rendered most homologous to the corresponding
string in an second natural antibody, a third resultant variant string in the variant antibody is
rendered most homologous to the corresponding string in an third natural antibody, the
substitutions are not in a CDR, and at least one resultant string is not a consensus of
homologous natural sequences. In an additional embodiment, the first, second and third
natural antibodies are from different subfamilies. If a further additional embodiment, the
variant antibody further comprises a fourth resultant variant string that is rendered most
homologous to the corresponding string in a fourth natural antibody. If an additional
embodiment, the variant antibody includes at least one substitution that is made at a position
that is not surface exposed, the first, second and third natural antibodies are from different
antibody groups, one of the substitutions is made at a position that is part of the VH/VL
interface, and at least one amino acid substitution is not a back mutation.
[162] In other embodiments, the first, second and third natural antibodies are from different
antibody groups.
[163] In other embodiments, the variant antibody includes at least one substitution that is
not surface exposed.
[164] In other embodiments, at least one of the substitutions is made at a position that is
part of the VH/VL interface.
[165] In other embodiments, the variant antibody includes at least one amino acid
substitution is not a back mutation.
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[166] In one embodiment of the present invention, a variant antibody for a host as
compared to a parent antibody includes a variant VH antibody region with host string content
(HSC) greater than about 75%, and a framework region homogeneity (FRH) less than about
60%, wherein the HSC and FRH are calculated with a window size of 9.
[167] In one embodiment of the present invention, a variant antibody for a host as
compared to a parent antibody includes a variant VH antibody region with exact string
content greater than about 20%; and a framework region homogeneity less than about 60%,
wherein the HSC and FRH are calculated with a window size of 9.
[168] In one embodiment of the present invention, a variant antibody for a host as
compared to a parent antibody includes a variant VL antibody region with exact string
content greater than about 35%, and a framework region homogeneity less than about 60%,
wherein HSC and FRH are calculated with a window size of 9.
[1 69] In one embodiment of the present invention, a variant antibody for a host as
compared to a parent antibody includes a first set of one or more amino acid substitutions
from a first natural antibody and a second set of one or more amino acid substitutions from a
second natural antibody, wherein the identity of said substituted amino acids from said
second antibody differ from the corresponding amino acids of said first natural antibody, the
substitutions are not in a CDR, and at least one substitution is not a consensus of
homologous natural sequences. In an additional embodiment, the variant antibody further
includes a third set of one or more amino acids substitutions from a third natural antibody
wherein the identity of the substituted amino acids of said third set differ from the identity the
corresponding amino acids from said first and second sets of amino acid substitutions. In a
further embodiment, the variant antibody includes a fourth set of one or more amino acids
from a fourth natural antibody wherein the identity of the substituted amino acids of said
fourth set differ from the identity the corresponding amino acids from said first, second and
third sets of amino acid substitutions. In other embodiments, the variant antibody includes
multiple sets of one or more amino acids from multiple natural antibodies, wherein the
identity of the substituted amino acids of any set drffer from the identity the corresponding
amino acids from the other sets of amino acid substitutions.
EXAMPLES
[170] Examples are provided below to illustrate the present invention. These examples are
not meant to constrain the present invention to any particular application or theory of
operation.
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[171] For reference to immunoglobulin variable regions, positions are numbered according
to the Kabat numbering scheme. For reference to immunoglobulin constant regions,
positions are numbered according to the EU index as in Kabat (Kabat et a/., 1991 ,
Sequences of Proteins of Immunological Interest, 5th Ed., United States Public Health
Service, National Institutes of Health, Bethesda).
Example 1. Immunoqenicity Reduction of AC10
[172] To illustrate application of the method described in the present invention, and to
validate its broad applicability to immunogenicity reduction of proteins, a xenogeneic
antibody example is provided using as the parent sequence the anti-CD30 antibody AC 10
(Bowen et a/. Journal of Immunology, 1993, 151: 5896). A structural model of the mouse
AC10 variable region was constructed using standard antibody modeling methods known in
the art. Figures 3 and 4 show the sequences, host string content, and structures of the
AC10 VL and VH domains (referred to as L0 AC10 VL and HO AC10 VH respectively). A
CDR graft of this antibody was constructed by placing the AC 10 CDRs into the context of the
frameworks of the most homologous host germlines, determined to be vlk_4-1 for VL and
vhJ-3 for VH using the sequence alignment program BLAST. The sequences and string
content of these CDR grafts are shown in Figures 5 and 6, along with structures of modeled
AC10 highlighting the mutational differences between the CDR grafted AC10 variable chains
and WT.
[173] AC10 variants with reduced immunogenicity were generated by applying a string
optimization algorithm on the WT AC1 0 VL and VH sequences. This algorithm heuristically
samples multiple amino acid mutations that exist in the diversity of the human VUc and VH
germline sequences, and calculates the host string content (HSC) of each sequence
according to Equation 3 described above, using a window size w=9. In this set of
calculations, residues in the CDRs and close to a CDR or to the VL/VH interface were
masked, that is were not allowed to mutate. CDRs were defined as a slightly smaller set of
residues than the CDRs defined by Chothia (Chothia & Lesk, 1987, J. Mol. Biol. 196: 901-
917; Chothia et a/., 1989, Nature 342: 877-883; Al-Lazikani etal. f 1997, J. Mol Biol. 273:
927-948). For the purposes of the present invention, VL CDRs are herein defined to include
residues at positions 27-32 (CDR1), 50-56 (CDR2), and 91-97 (CDR3), wherein the
numbering is according to Chothia. Because the VL CDRs as defined by Chothia and Kabat
are identical, the numbering of these VL CDR positions is also according to Kabat. For the
purposes of the present invention, VH CDRs are herein defined to include residues at
positions 27-33 (CDR1), 52-56 (CDR2), and 95-102 (CDR3), wherein the numbering is
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according to Chothia. These VH CDR positions correspond to Kabat positions 27-35
(CDR1), 52-56 (CDR2), and 95-102 (CDR3). Masked residues in these calculations were
set at positions 1-4, 25-34, 36, 38, 43, 44, 46, 48-58, 60, 63-69, 71 , 87, and 89-98 for VL,
and 2, 4, 24, 26-35, 37, 39, 44, 45, 47, 50-58, 60, 61, 71, 73, 76, 78, 91, and 93-106 for VH,
wherein the numbering is according to Kabat Masking of potentially critical residues is a
conservative approach to generating more host antibody variants, however it is but one
embodiment of the present invention, and calculations wherein positions are not masked are
also contemplated. This calculation was run for AC10 VL and VH in 100 separate
interactions, generating a set of diverse AC 10 variants with more host string content than
WT. Figure 7 shows the clustered nonredundant set of output sequences from these
calculations for the AC10 VL and VH region, referred to as AC10 VL HSC Calculation 1 and
AC10 VH HSC Calculation 1 respecitvely. For each iteration (Iter), the HSC (Eqation 3),
HSS (Equation 5), and number (Mut) and identity (shaded residues) of mutations from WT
are presented. In addition to the HSC score, each sequence was evaluated for its structural
and functional integrity using a nearest neighbor structure-based scoring method (USSN
60/528,229, filed December 8, 2003, entitled Protein Engineering with Analogous Contact
Environments). Two measures of structural fitness, referred to as "Structural Consensus"
and "Structural Precedence", are also provided in the Figure 7. Although the Analogous
Contact Environments method is particularly well-suited for antibodies because of the wealth
of sequence and structure information, any structure-based and/or sequence-based scoring
method may be used to evaluate the structural and functional fitness of the variant
sequences. The output sequences were clustered based on their mutational distance from
the other sequences in the set, and these clusters are delineated by the horizontal black
lines in the Figure. The "Cluster" column provides the quantitative mutational distance
between each sequence and the rest of the sequence in its cluster; sequences with a lower
cluster value are more representative of that particular sequence cluster.
[174] These calculations were used to generate a set of AC10 VL and VH variants. In
some cases, further substitutions were made to output sequences, using string and
structural scores, as well as visual inspection of the modeled AC10 structure, to evaluate
fitness. Figures 8-13 present the sequences, host string content, and mapped mutational
differences on the modeled AC 10 structure for each of the AC 10 VL and VH variants.
Iteration 36 from AC10 VL HSC calculation 1 served as the precursor for L1 AC10 VL,
iteration 37 served as the precursor for L2 AC 10 VL, and iteration 3 served as the precursor
for L3 AC10 VL. Iteration 15 from AC10 VH HSC calculation 1 served as the precursor for
H1 AC 10 HL, iteration 55 served as the precursor for H2 AC 10 VH, and iteration 18 served
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as the precursor for H3 AC10 VH.
[175] Tables 1 and 2 present the number of mutations from the parent sequence, structural
fitness scores, and host string scores for the AC10 VL and VH variants as compared to the
WT and CDR grafted AC10 sequences. In addition to the aforementioned structural and
host string analysis, each sequence was analyzed for its global homology to the host
germline. The maximum identity match to the germline for each string in the sequences was
also determined, referred to as N| D max. This represents the total number of strings in each
sequence whose maximum identity to the corresponding strings in the host germline is the
indicated value. For w = 9, Tables 1 and 2 list N 9 max, N 8 max, N 7 max, and N^max for each
sequence. N 9 max represents the number of strings in the sequence for which 9 of 9
residues match at least one string in the host germline, N 8 max represents the number of
strings for which 8 of 9 residues match at least one string in the host germline, N 7 max
represents the number of strings for which 7 of 9 residues match at least one string in the
host germline, and N^max represents the number of strings for which 6 or less residues of 9
residues match at least one string in the host germline. This last category (ID <6) could, for
example, be regarded as the number of poorly scoring strings. In addition to the
aforementioned structural and host string analysis, each sequence was analyzed for its
global homology to the host germline; Tables 1 and 2 present the most homologous human
germline sequence for each sequence (Closest Germline) and corresponding identity to that
germline (ID to Closest Germline), determined using the sequence alignment program
BLAST. Finally, the Framework region homogeneity (FRH) of each variant was evaluated
for w=9, and is presented in Tables 1 and 2, providing the extent to which the host string
content of each variant is derived from a single germline as opposed to multiple germline
sequences.
Table 1. AC10 VL Variants
L1 L2 L3
15 16 9
0.59 0.64 0.56
0.66 0.67 0.58
0.86 0.86 0.85
0.48 0.46 0.41
WT CDR
Wl Graft
Mutations 18
Structural
Consensus
Structural
Precedence
Human String
Content
Human String
Similarity
0.57 0.57
0.68 0.57
0.78 0.88
0.15 0.57
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WT
CDR
Graft
Framework Region
Homogeneity
N 9 max 15 61
N 8 max 27 11
N 7 max 31 15
N^max 34 20
4-1 4-1
L1
L2
L3
0.73
0.81
0.52
51
48
42
13
15
21
21
22
20
22
22
24
3-11
1-39
4-1
Closest
Germline
ID to Closest 68/101 867 101 78 / 99 80 / 99 75/101
Germline 67% 85% 79% 81% 74%
Table 2. AC10 VH Variants
WT
CDR
Graft
H1
H2
H3
Mutations
26
16
23
20
Structural
0.49
0.48
0.48
Consensus
0.50
0.47
Structural
0.63
0.67
0.63
Precedence
0.59
0.59
Human String
0.69
0.87
0.81
Content
0.81
0.80
Human String
Similarity
0.07
0.68
0.41
0.39
0.38
Framework
Region
0.60
0.86
0.65
0.47
0.55
Homogeneity
N 9 max
5
81
48
45
44
N 8 max
31
13
30
32
28
N 7 max
34
8
20
21
24
N^max
49
17
21
21
23
Closest
1-3
1-3
1-3
Germline
1-3
7-4-1
ID to Closest
69 / 98 93 / 98 83 198 721 98 76 / 9£
Germline
70%
95%
85%
73%
78%
[176] An important observation is that, whereas the CDR grafted antibodies are most
homologous to a single human germline sequence (the "acceptor" sequence in humanization
terminology), the present invention describes variants that are homologous to different host
germline sequences in different regions of the sequence. This is evident from the significant
differences in Framework region homogeneity (FRH) scores for the AC 10 variants of the
present invention and CDR grafted AC10 variants. Furthermore, whereas CDR grafted
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AC10 VL and VH are most homologous to human germline subfamilies 4 (VL) and 1 (VH)
respectively across their entire sequences, a number of the AC10 variants are most
homologous to different subfamilies in different frameworks. Additionally, whereas the CDR
grafted antibodies are most homologous to a single germline sequence that is also the most
homologous sequence to the parent sequence, the present invention presents a set of
antibodies for a given antibody that are most homologous to different human germline
sequences, which need not be the most homologous germline sequence to WT. For
example, Table 1 shows that CDR grafted AC10 VL is most homologous to 4-1 , which is
also the most homologous human germline to the WT AC10 parent. However L1, L2, and
L3 are most homologous to three different human germlines -3-11, 1-39, and 4-1
respectively. Thus the variants of the present invention explore a substantially greater
amount of diversity than CDR grafted antibodies. One obvious advantage of this is that the
method of the present invention provides a greater chance of success with respect to
antigen affinity. The choice of an "acceptor" in humanization methods places a single bet; if
the donor CDRs are in fact incompatible with the acceptor FRs, a set of backmutations that
regain WT affinity may not exist. In contrast, the method of the present invention enables a
greater diversity of sequence and structure space to be sampled in the immunogenicity
reduction process, increasing the chances of obtaining a final less immunogenic version with
WT affinity or better. An additional advantage of sampling greater sequence diversity is that
some sequences may have more optimal properties than others, for example with regard to
stability, solubility, and effector function. For example, as disclosed in USSN 60/614,944,
and USSN 60/619,409, filed October 14, 2004, entitled "Immunoglobulin Variants Outside
the Fc Region with Optimized Effector Function", the variable region of an antibody may
impact effector functions such as antibody dependent cell-mediated cytotoxicity (ADCC),
antibody dependent cell-mediated phagocytosis (ADCP), and complement dependent
cytotoxicity (CDC).
[177] The genes for the variable regions of AC1 0 WT (L0 and HO) and variants (L1 , L2, L3,
H1, H2, and H3) were constructed using recursive PCR, and subcloned into a the
mammalian expression vector pcDNA3.1Zeo (Invitrogen) comprising the full length light
kappa (CU) and heavy chain lgG1 constant regions. All sequences were sequenced to
confirm the fidelity of the sequence. Plasmids containing heavy chain gene (VH-CH1-CH2-
CH3) (wild-type or variants) were co-transfected with plasmid containing light chain gene
(VL-CLO) in all combinations (L0/H0, L0/H1, L0/H2, L0/H3, L1/H0, L1/H1, L1/H2, L1/H3,
L2/H0, L2/H1 , L27H2, L2/H3, L3/H0, L3/H1 , L3/H2, L3/H3) into 293T cells. Here, for
example, L2/H3 refers to the L2 AC10 VL paired with H3 AC10 VH. Media were harvested 5
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days after transfection, and antibodies were purified from the supernatant using protein A
affinity chromatography (Pierce, Catalog # 20334).
[178] WT and variant antibodies were experimentally tested for their capacity to bind CD30
antigen. Binding affinity to human CD30 by the AC10 WT and variant antibodies was
measured using a quantitative and extremely sensitive method, AlphaScreen™ assay. The
AlphaScreen™ assay is a bead-based non-radioactive luminescent proximity assay. Laser
excitation of a donor bead excites oxygen, which if sufficiently close to the acceptor bead will
generate a cascade of chemiluminescent events, ultimately leading to fluorescence emission
at 520-620 nm. The AlphaScreen™ assay was applied as a competition assay for screening
the antibodies. WT AC10 antibody was biotinylated by standard methods for attachment to
streptavidin donor beads (Perkin Elmer). Commericial CD30 was conjugated to digoxigenin
(DIG) (Roche Diagnostics) for attachment to anti-DIG acceptor beads (Perkin Elmer). In the
absence of competing AC10 variants, WT antibody and CD30 interact and produce a signal
at 520-620 nm. Addition of untagged ACiO variant competes with the WT AC10 / CD30
interaction, reducing fluorescence quantitatively to enable determination of relative binding
affinities. Figures 14a and 14b show binding of WT (H0L0) and AC10 variant antibodies to
CD30 using the AlphaScreen™ assay. The data were fit to a one site competition model
using nonlinear regression, and these fits are represented by the curves in the figure. These
fits provide the inhibitory concentration 50% (IC50) (i.e. the concentration required for 50%
inhibition) for each antibody, thus enabling the relative binding affinities relative to WT to be
determined. Table 3 provides the IC50's and Fold lC50's relative to WT for fits to these
binding curves. The AC10 variants display an array of CD30 binding affinities, with a
number of variants binding CD30 with affinity comparable to or better affinity than WT AC10.
[179] Antigen affinity of the AC10 variants was also measured using Surface Plasmon
Resonance (SPR) (Biacore, Uppsala, Sweden). SPR allows for the measurement of direct
binding rates and affinities of protein-protein interactions, and thus provides an excellent
complementary binding assay to the AlphaScreen™ assay. CD30 fused to the Fc region of
lgG1 (R&D Systems) was immobilized on a Protein A SPR chip, the surface was blocked
with Fc, and WT and variant AC 10 antibodies were flowed over the chip at a range of
concentrations. The resulting sensorgrams are shown in Figure 15. Global Langmuirfrts
were carried out for the concentrations series using the BiaEvaluation curve fitting software,
providing the on-rate constant (ka), off-rate constant (kd), and equilibrium binding constant
(KD=kd/ka) for the curves. Table 3 provides the KDs and Fold KDs relative to WT for the
SPR data. The excellent agreement between the rank ordering of the variants as
determined by SPR and AlphaScreen™ assay support the accuracy of the data.
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Table 3. CD30 Binding of AC10 Variants
AC10
Variant
SPR
KD (nM)
SPR
Fold KD
AlphaScreen
IC50(nM)
AlphaScreen
Fold IC50
H2L1
9.49
0.36
55.1
0.06
H2L2
5.95
0.57
49.2
0.06
H1L2
7.55
0.45
45.3
0.07
H1L1
5.63
0.60
27.7
0.11
H2L3
6.75
0.50
27.2
0.12
H2L0
8.00
0.42
19.4
0.16
H1L3
5.09
0.67
17.4
0.18
H1L0
6.39
0.53
9.77
0.32
H0L2
3.48
0.97
7.81
0.41
H3L2
2.86
1.19
6.57
0.48
H3L0
3.08
1.10
6.18
0.51
H3L1
2.44
1.39
6.09
0.52
H0L1
3.29
1.03
5.19
0.61
H0L3
3.00
1.13
4.61
0.69
H0LO
3.39
1.00
3.18
1.00
H3L3
2.33
1.45
1.99
1.59
[180] In addition to assessing the antigen affinity and biophysical properties of the variants
of the present invention, they may also be tested for effector functions in the context of a full
length antibody. One advantage of generating multiple reduced immunogenicity variants of
a parent immunoglobulin is that it enables a greater degree of sequence diversity to be
sampled, diversity which may provide optimal properties. Some sequences may have more
optimal properties than others, for example with regard to effector function. For example, as
disclosed in USSN 60/614,944, and USSN 60/619,409, filed October 14, 2004, entitled
"Immunoglobulin Variants Outside the Fc Region with Optimized Effector Function", the
variable region of an antibody may impact effector functions such as antibody dependent
cell-mediated cytotoxicity (ADCC), antibody dependent cell-mediated phagocytosis (ADCP),
and complement dependent cytotoxicity (CDC).
[181] In order to explore any differences in capacity to mediate effector function, the
affinities of the AC10 variants for FcKRIIIa were measured using the AlphaScreen™ assay.
The extracellular region of human V158 F^RHIa was obtained by PCR from a clone
obtained from the Mammalian Gene Collection (MGC:22630), and the receptor was fused
with glutathione S-Transferase (GST) to enable screening. Tagged FcKRIIIa was
transfected in 293T cells, and media containing secreted FcKRIIIa were harvested and
purified. The AlphaScreen™ assay was applied as a competition assay for screening AC10
variants for binding to FcKRIIIa. Biotinylated WT AC10 antibody was bound to streptavidin
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donor beads (Perkin Elmer), and GST-fused human V158 F^RIHa was bound to anti-GST
acceptor beads (Perkin Elmer). The binding data are shown in Figures 16a and 16b, and
the resulting IC50's and Fold IC50's relative to WT are provided in Table 4. FcKRIIIa affinity
of the AC10 variants was also measured using SPR. GST-fused human FcKRIIIa (V158
isoform) was immobilized on a chip, and WT and variant AC10 antibodies were flowed over
the chip at a range of concentrations. Binding constants were obtained from fitting the data
using standard curve-fitting methods, The equilibrium dissociation constants (KDs) obtained
from the fits to these binding curves, and the calculated fold improvement or reduction
relative to WT (Fold KD) are shown in Table 4.
Table 4. FcKRIIIa Binding of AC10 Variants
AC10
Variant
SPR
KD {nM)
SPR
Fold KD
AlphaScreen
IC50 (nM)
AlphaScreen
Fold IC50
H2L1
14.9
1.25
751
0.12
H2L2
4.01
4.64
146
0.60
H1L2
1.6.6
1.12
340
0.26
H1L1
11.2
1.66
221
0.39
H2L3
3.52
5.28
183
0.48
H2L0
12.9
1.44
175
0.50
H1L3
11.2
1.66
178
0.49
H1L0
22.0
0.85
71.6
1.22
H0L2
9.09
2.05
93.8
0.93
H3L2
3.57
5.21
88.7
0.98
H3L0
20.0
0.93
216
0.40
H3L1
17.4
1.07
209
0.42
H0L1
11.6
1.60
183
0.48
H0L3
12.7 j
1.46
146
0.60
H0L0
18.6
1.00
87.2
1.00
H3L3
6.13
3.03
83.5
1.04
[182] To assess the capacity of the AC10 variants to mediate effector function against
CD30 expressing cells, the AC 10 variants were tested in a cell-based ADCC assay. Human
peripheral blood monocytes (PBMCs) were isolated from buffy-coat and used as effector
cells, and CD30 positive L540 Hodgkin's lymphoma cells were used as target cells. L540
target cells were seeded at 20,000 per well in 96-welI plates and treated with designated
antibodies in triplicates starting at 1 pg/ml and in reduced concentrations in 34 log steps.
PBMCs isolated using a Ficoll gradient and allotyped as FcKRIIIa 158 V/F were added at 25-
fold excess of L540 cells and co-cultured for 4 hrs before processing for LDH activity using
the Cytotoxicity Detection Kit (LDH, Roche Diagnostic Corporation, Indianapolis, IN)
according to the manufacturer's instructions. The plates were read using a Wallac 1420
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Victor 2 ™. Figures 17a - 17c show the results. The graphs show that the antibodies differ
not only in their EC50, reflecting their relative potency, but also in the maximal level of ADCC
attainable by the antibodies at saturating concentrations, reflecting their relative efficacy.
These two terms, potency and efficacy, are sometimes used loosely to refer to desired
clinical properties. In the current experimental context, however, they are denoted as
specific quantities, and therefore are here explicitly defined. By " potency" as used in the
current experimental context is meant the EC50 of an EGFR targeting protein. By " efficacy"
as used in the current experimental context is meant the maximal possible effector function
of an antibody at saturating levels. Differences in capacity to mediate ADCC may be due to
differences in antigen affinity, different capacities of the variant variable regions to effect
FckR binding, or both. Regardless, the contribution of an antibody variable region to Fc/R
binding and effector function may be an important parameter for selecting a clinical
candidate. The choice of an antibody clinical candidate based in whole or in part on the
impact on effector function of the variable region represents a novel dimension in antibody
therapeutics.
[183] Based on the CD30 binding, FcKRIIIa binding, and ADCC results, the H3/L3 AC10
variant was chosen as a potential biotherapeutic candidate. Because this antibody is
intended for clinical use as an anti-cancer therapeutic, it may be advantageous to optimize
its effector function. As previously described, substitutions can be engineered in the
constant region of an antibody to provide favorable clinical properties. In a most preferred
embodiment, one or more amino acid modifications that provide optimized binding to Fc/Rs
and/or enhanced effector function described in USSN 10/672,280, PCT US03/30249, and
USSN 10/822,231, and USSN 60/627,774, filed 11/12/2004 and entitled "Optimized Fc
Variants", are combined with the AC10 variants of the present invention. A number of
optimized Fc variants obtained from these studies, including I332E, S239D, V264I/I332E,
S239D/I332E, and S239D/A330L/I332E, were constructed in the H0/L0 and H3/L0 AC10
antibodies using quick change mutagenesis (Stratagene). Antibodies were expressed and
purified as described above. Figures 18a and 18b show the results of the ADCC assay,
carried out as described above, comparing WT (H0/L0) and H3/L3 AC10 in combination with
the optimized Fc variants. Considerable enhancements in potency and efficacy are
observed for the Fc variant antibodies as compared to H0/L0 and H3/L3 AC 10.
[184] As described above, because variant sequences of the invention are preferably
derived from a HSC-increasing procedure in which substitution of structurally important
positions is disallowed (or discouraged), it is likely that additional optimization of HSC is
possible if those positions are allowed to vary in a secondary analysis. It is noted that, due
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PCTAJS2004/040694
to residue masking, mutations in the variants occur distal to the CDRs and VL/VH interface.
This is in contrast to CDR grafted antibodies, which have mutations in the parent that are at
antigen affinity. This is corroborated by the fact that CDR grafted antibodies typically require
backmutations to the donor sequence to regain WT affinity for antigen. Such backmutations
are usually made out of structural and immunogenic context with respect to host sequences,
and cause dramatic reductions in the host string content of the final variant. In contrast, the
variants presented herein are simultaneously optimized for host string and structural fitness
within the same context, and no backmutations need be made. Nonetheless, one or more
subsequent substitutions may be explored to increase antigen affinity or further improve
HSC, for example by mutating residues that were masked in the calculations and/or residues
in or close to the CDRs or VL/VH interface. Thus the H3/L3 variant can be thought of as a
primary variant or template for further optimization, and variants of H3/L3 can be thought of
as secondary variants. In contrast to backmutating as with CDR grafted antibodies,
secondary substitutions in the variants of the present invention will comprise forward or
neutral mutations with respect to the host germline, and thus are expected to only improve or
unaffected HSC. An additional benefit of generating secondary variants is that, by exploring
quality structural and string diversity, it is also possible that other properties can be
optimized, for example affinity, activity, specificity, solubility, expression level, and effector
[185] String analysis was carried out on the H3/L3 sequence to design a set of secondary
substitutions that have neutral, positive, or minimal impact on HSC, and/or that have
significant potential for optimization of antigen affinity and/or effector function. Table 5
provides this set of 70 VL (Table 5a) and 64 VH (Table 5b) single mutations. The H3 column
provides the WT H3 amino acid, and the Sub column provides the designed substitution.
Positions are numbered according to the Kabat numbering format, with Kabat CDR positions
bolded. The provided string impact defined according to Equation 7, describes the
difference in HSC between the primary variant sequence, here H3/L3, and the secondary
variant sequence.
or near these critical regions and thus have a significantly greater potential for perturbing
function.
Table 5a. L3 AC10 Secondary Variants
Variant
Pos
(Kabat)
L3 Sub
String Fold Fold Fold
Impact ProtA CD30 FcrRMIa
L3.1
L3.2
1
1
D
D
A
E
0
1
0.89
1.09
1.30
1.24
0.96
1.46
53
WO 2005/056759
Variant
L3.3
L3.4
L3.5
L3.6
L3.7
L3.8
L3.9
L3.10
L3.11
L3.12
L3.13
L3.14
L3.15
L3.16
L3.17
L3.18
L3.19
L3.20
L3.21
L3.22
L3.23
L3.24
L3.25
L3.26
L3.27
L3.28
L3.29
L3.30
L3.31
L3.32
L3.33
L3.34
L3.35
L3.36
L3.37
L3.38
L3.39
L3.40
L3.41
L3.42
L3.43
L3.44
L3.45
L3.46
L3.47
L3.48
L3.49
L3.50
PCT/US2004/040694
Pos
L3
Sub
String
Fold
Fold
Fold
(Kabat)
Impact
ProtA
CD30
FcKRIIIa
1
D
N
0
1.24
1.66
1.35
1
D
S
0
0.97
1.04
1.18
3
V
Q
0
1.13
1.32
1.32
4
L
M
.4
1.65
1.64
1.21
25
A
S
6
27a
S
D
0
1.02
0.94
0.61
27b
V
I
1
1.04
0.58
0.81
27c
D
L
5
27c
D
S
8
27c
D
V
3
1.24
1.15
1.19
27d
F
D
0
1.07
0.32
0.98
27d
F
H
4
0.86
0.08
0.93
27d
F
Y
5
i.04
0.63
1.19
28
D
N
-1
1.10
1.61
1.18
30
D
K
4
1.01
1.21
1.24
30
D
N
6
1.09
1.45
0.94
30
D
S
4
1.07
1.13
0.82
30
D
Y
0
0.82
0.78
0.73
31
S
D
0
1.01
0.81
0.95
31
S
T
3
1.03
0.46
0.97
31
S
N
-1
1.03
0.71
1.00
32
Y
D
0
1.31
0.46
1.33
33
M
L
8
1.38
1.36
1.37
34
N
S
0
34
N
A
-1
1.39
0.38
1.36
34
N
D
-6
1.19
0.41
1.76
46
V
H
4
0.06
0.03
0.11
46
V
L
9
0.86
0.39
0.75
46
V
R
4
46
V
S
4
1.05
0.32
0.90
50
A
D
6
0.98
0.26
0.66
50
A
S
5
1.01
0.47
1.20
50
A
W
2
53
N
s
8
53
N
T
5
0.99
1.26
1.01
54
L
R
1
1.19
1.46
1.55
55
E
A
2
1.01
0.85
1.32
55
E
Q
6
0.99
0.87
1.07
56
S
T
8
1.50
1.80
1.23
58
I
V
4
1.44
1.55
0.95
60
A
D
1
1.11
1.16
1.08
60
A
S
2
0.82
1.08
0.85
67
S
P
0
89
Q
H
1
1.37
0.08
1.64
91
S
A
8
91
S
G
9
0.85
0.29
0.80
91
S
H
2
.1.20
0.01
1.32
91
S
L
8
1.10
0.02
1.59
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Variant
Pos
(Kabat)
L3
Sub , Strin 9
Impact
Fold
Prot A
Fold
CD30
Fold
FcKRIHa
L3.51
91
s
Y
8
1.00
0.02
1.50
L3.52
92
N
I
3
L3.53
92
N
S
2
3.02
0.48
1.34
L3.54
92
N
Y
8
1.39
0.96
1.05
L3.55
93
E
K
8
0.62
0.27
0.49
L3.56
93
' E
N
8
1.06
0,64
0.84
L3.57
93
E
Q
2
L3.58
93
E
S
8
0.90
0.49
0.87
L3.59
94
D
A
3
1.16
0.09
1.14
L3.60
94
D
F
9
1.22
0.02
1.19
L3.61
94
D
H
8
L3.62
94
D
L
3
0.87
0.46
0.79
L3.63
94
D
S
1
1.74
0.57
1.42
L3.64
94
D
T
7
1.24
0.14
1.16
L3.65
96
W
F
0.33
0.34
0.29
L3.66
96
W
I
0.75
0.00
0.57
L3.67
96
w
L
L3.68
96
w
Y
L3.69
100
G
P
L3.70
100
G
Q
Table 5b. H3 AC10 Secondary Variants
Variant
Position
(Kabat)
H3
Sub
String
Impact
Fold
Prot A
Fold
CD30
H3.1
1
Q
E
-1
0.83
1.00
H3.2
2
I
L
0
1.60
2.76
H3.3
2
I
M
2
0.88
0.68
H3.4
2
I
V
0
0.98
1.28
H3.5
9
P
A
2
0.95
1.29
H3.6
16
A
T
2
0.89
1.13
H3.7
24
A
V
2
1.54
4.45
H3.8
31
D
G
2
0.80
1.40
H3.9
31
D
S
2
0.82
1.65
H3.10
33
Y
D
2
0.68
0.07
H3.11
33
Y
G
3
0.96
0.73
H3.12
33
Y
W
0
0.84
0.00
H3.13
34
I
L
1
0.96
1.52
H3.14
34
I
M
8
1.05
1.62
H3.15
35
T
D
1
1.55
0.05
H3.16
35
T
G
2
1.03
0.15
H3.17
35
T
H
8
0.86
0.04
H3.18
35
T
N
4
1.07
0.13
H3.19
35
T
S
6
0.88
1.11
H3.20
44
G
A
0
1.20
2.04
H3.21
44
G
R
0
1.36
2.60
H3.22
50
W
I
6
1.25
0.01
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Variant
Position
H3
Sub
String
Fold
Fold
(Kabat)
impact
ProtA
CD30
H3.23
50
W
R
0
0.99
0.16
H3.24
52
Y
N
2
1.03
0.03
H3.25
52
Y
T
•j
1.11
0.06
H3.26
52
Y
V
•j
1.33
0.06
H3.27
52a
P
A
1.02
2.00
H3.28
52a
P
V
•j
1.44
1.34
H3.29
54
S
D
1.45
1.81
H3.30
54
S
N
5
1.13
1.45
H3.31
58
K
G
4
H3.32
58
K
I
2
1.22
1.09
H3.33
58
K
N
5
1.26
0.50
H3.34
60
N
A
7
0.87
1 30
H3.35
60
N
P
o
H3.36
60
N
S
7
1.02
1.24
H3.37
60
N
T
0
1.12
1.01
H3.38
60
N
V
0
1.16
1.14
H3.39
60
N
D
0
1.09
1.00
H3.40
61
E
Q
7
1.51
1.83
H3.41
64
Q
T
0
0.98
1.38
H3.42
71
V
L
4
1.10
0.66
H3.43
71
V
M
9
1.17
0.88
H3.44
71
V
R
1
1.25
1.76
H3.45
87
T
M
-1
0.99
1.14
H3.46
89
V
M
-4
1.41
1.39
H3.47
91
F
H
2
H3.48
91
F
Y
9
1.32
1.60
H3.49
93
A
T
0
1.47
0.38
H3.50
93
A
V
0
1.01
1 40
H3.51
94
N
A
9
1.51
0.08
H3.52
94
N
H
5
1.23
1.24
H3.53
94
N
K
9
1.67
0 02
H3.54
94
N
R
9
1.26
0.00
H3.55
94
N
T
g
1.24
0 91
H3.56
99
W
Y
1.26
0.07
H3.57
101
A
D
1.31
0.53
H3.58
101
A
Q
1.17
0.16
H3.59
102
Y
H
1.69
1.05
H3.60
102
Y
S
1.04
0.82
H3.61
102
Y
V
1.33
1.21
H3.62
102
Y
L
1.34
1.22
H3.63
102
Y
F
1.18
1.24
H3.64
105
Q
R
1.15
1.28
[186] The secondary. H3/L3 variants were constructed using quick change mutagenesis,
and the full length antibodies were expressed and purified as described above. H3 variants
comprised H3 variant VH chains(H3.1- H3.64) in combination with L3 VL, and L3 variants
comprised L3 variant VL chains (L3.1 ~ L3.70) in combination with H3 VH. The
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AlphaScreen™ assay was used to measure binding of the H3/L3 secondary variants to
CD30 and FcKRIHa (as described earlier), as well as to protein A using biotinylated AC 10
bound directly to protein A acceptor beads and streptavidin donor beads. Figure 19 provides
AlphaScreen™ binding curves for binding of select AC10 variants to CD30. The Fold IC50's
relative to WT H3/L3 for binding to CD30, FcKRIHa, and protein A are provided in Table 5. A
number of H3/L3 secondary variants provide comparable or improved binding to CD30
antigen relative to the H3/L3 parent, enabling the engineering of additional variants that
comprise combinations of these substitutions, which may provide further enhancements in
HSC and/or antigen affinity.
[187] Secondary substitutions that show favorable properties with respect to antigen
affinity, effector function, stability, solubility, expression, and the like, may be combined in
subsequent variants to generate a more optimized therapeutic candidate. Two new VL and
three new VH variants were designed that comprise combinations of the described
secondary substitutions, referred to as L3.71, L3 .72, H3.68, H3.69, and H3.70. Figures 20 -
24 present the sequences, host string content, and mapped mutational differences on the
modeled AC10 structure for each of these new AC10 VL and VH variants. Table 6 presents
the number of mutations from the parent sequence, structural fitness scores, host string
scores, and homology scores for these AC 10 VL and VH variants.
Table 6. AC10 Variants
L3.71
L3.72
H3.68
H3.69
H3.70
Mutations
15
15
23
27
30
Structural
0.56
0.55
0.46
Consensus
0.46
0.45
Structural
0.54
0.52
Precedence
0.55
0.57
0.56
Human
String
0.88
0.87
0.80
0.83
0.84
Content
Human
String
0.52
0.45
0.39
0.47
0.47
Similarity
Framework
Region
0.47
0.51
0.33
0.40
0.42
Homogeneity
N 9 max
55
47
46
55
55
N 8 max
19
24
26
26
34
N 7 max
14
17
24
20
12
N^max
19
19
23
18
18
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L3.71
L3.72
H3.68
H3.69
H3.70
Closest
Germline
4-1
4-1
7-4-1
1-3
1-3
ID to Closest
76/101
76/101
74/98
77/98
79/98
Germline
75%
75%
76%
79%
81%
[188] Because the provided AC 10 variants antibodies are clinical candidates for anti-
cancer therapeutics, it may be advantageous to optimize their effector function. As
previously described, substitutions can be engineered in the constant region of an antibody
to provide favorable clinical properties. Combinations of the variants of the present invention
with Fc modifications that alter effector function are anticipated. In a most preferred
embodiment, one or more amino acid modifications that provide optimized binding to FcyRs
and/or enhanced effector function described in USSN 10/672,280, PCT US03/30249, and
USSN 10/822,231, and USSN 60/627,774, filed 11/12/2004 and entitled "Optimized Fc
Variants", are combined with the AC10 variants of the present invention. The optimal antt-
CD30 clinical candidate may comprise amino acid modifications that reduce immunogenicity
and enhance effector function relative to a parent anti-CD30 antibody. Figures 25a - 25c
provide the light and heavy chain sequences of AC10 variants that comprise L3.71/H3.70
AC10 as described above, combined with a number of possible variant lgG1 constant
regions, comprising one or more modifications at S239, V264, A330, and I332, that provide
enhanced effector function.
[189] Although human lgG1 is the most commonly used constant region for therapeutic
antibodies, other embodiments may utilize constant regions or variants thereof of other IgG
immunoglobulin chains. Effector functions such as ADCC, ADCP, CDC, and serum half-life
differ significantly between the different classes of antibodies, including for example human
lgG1, lgG2, lgG3, lgG4, lgA1, lgA2, IgD, IgE, IgG, and IgM (Michaelsen et al. t 1992,
Molecular Immunology, 29(3): 319-326). A number of studies have explored lgG1, lgG2,
lgG3, and lgG4 variants in order to investigate the determinants of the effector function
differences between them. See for example Canfield & Morrison, 1991, J. Exp. Med. 173:
1483-1491; Chappel et al., 1991, Proc. Natl. Acad. Sci. USA 88(20): 9036-9040; Chappel et
al., 1993, Journal of Biological Chemistry 268:25124-25131; Tao et al., 1991, J . Exp. Med.
173: 1025-1028; Tao et al., 1993, J. Exp. Med. 178: 661-667; Redpath et al., 1998, Human
Immunology, 59, 720-727. Using methods known in the art, it is possible to determine
corresponding or equivalent residues in proteins that have significant sequence or structural
homology with each other. By the same token, it is possible to use such methods to
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WO 2005/056759 PCT/US2004/040694
engineer amino acid modifications in an antibody or Fc fusion that comprise constant regions
from other immunoglobulin classes, for example as described in USSN 60/621,387 and
60/629,068, to provide optimal properties. As an example, the relatively poor effector
function of lgG2 may be improved by replacing key FcyR binding residues with the
corresponding amino acids in an IgG with better effector function, for example lgG1 . For
example, key residue differences between lgG2 and IgGI with respect to FcyR binding may
include P233, V234, A235, -236 (referring to a deletion in lgG2 relative to lgG1), and G327.
Thus one or more amino acid modifications in the parent lgG2 wherein one or more of these
residues is replaced with the corresponding lgG1 amino acids, P233E, V234L, A235L, -
236G (referring to an insertion of a glycine at position 236), and G327A, may provide
enhanced effector function. Furthermore, one or more additional amino acid modifications,
for example the S239D, V264I, A330L, I332E, or combinations thereof as described above,
may provide enhanced FcyR binding and effector function relative to the parent lgG2.
Figures 25a, 25d, and 25e illustrate this embodiment, providing the light and heavy chain
sequences of AC1 0 variants that comprise L3.71/H3.70 AC10 combined with a number of
possible variant lgG2 constant regions.
[190] The Fc modifications defined in Figure 25 that provide enhanced effector function are
not meant to constrain the invention to only these modifications for effector function
optimization. For example, as described in US 6,737,056, PCT US2004/000643, USSN
10/370,749, and PCT/US2004/005112, the substitutions S298A, S298D, K326E, K326D,
E333A, K334A, and P396L provide optimized FcyR binding and/or enhanced ADCC.
Furthermore, as disclosed in Idusogie et al., 2001, J. Immunology 166:2571-2572,
substitutions K326W, K326Y, and E333S provide enhanced binding to the complement
protein C1q and enhanced CDC. As described in Hinton et al., 2004, J. Biol. Chem. 279(8):
6213-6216, substitutions T250Q, T250E, M428L, and M428F provide enhanced binding to
FcRn and improved pharmacokinetics. Modifications need not be restricted to the Fc region.
It is also possible that the mutational differences in the Fab and hinge regions may provide
optimized FcyR and/or C1q binding and/or effector function. For example, as disclosed in
USSN 60/614,944, and USSN 60/619,409, filed October 14, 2004, entitled "Immunoglobulin
Variants Outside the Fc Region with Optimized Effector Function", the Fab and hinge
regions of an antibody may impact effector functions such as antibody dependent cell-
mediated cytotoxicity (ADCC), antibody dependent cell-mediated phagocytosis (ADCP), and
complement dependent cytotoxicity (CDC). Thus immunoglobulin variants comprising
substitutions in the Fc, Fab, and/or hinge regions are contemplated. For example, the
antibodies may be combined with one or more substitutions in the VL, CL, VH, CH1, and/or
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{
hinge regions. Furthermore, further modifications may be made in non lgG1
immunoglobulins to corresponding amino acids in other immunoglobulin classes to provide
more optimal properties, as described in USSN 60/621,387, filed 10/21/2004, entitled "IgG
Immunoglobulin Variants with Optimized Effector Function". For example, in one
embodiment, an lgG2 antibody, similar to the antibody presented in Figure 25, may comprise
one or more modifications to corresponding amino acids in lgG1 or lgG3 CH1, hinge, CH2,
and/or CH3. In another embodiment, an lgG2 antibody, similar to the antibody presented in
Figure 25, may comprise all of the lgG1 CH1 and hinge substitutions,, i.e. said lgG2 variant
comprises the entire CH1 domain and hinge of lgG1.
Example 2. Immunogenicitv Reduction of C225
[191] To illustrate further application of the method described in the present invention, and
to validate its broad applicability to immunogenicity reduction of proteins, a second
xenogeneic antibody example is provided using the variable region of C225 as the parent
sequence (cetuximab, Erbitux®, Imclone) (US 4,943,533; PCT WO 96/40210). C225 is a
murine anti-EGFR antibody, a chimeric version of which is currently approved for the
treatment of cancer. A structural model of the murine C225 variable region was constructed
using standard antibody modeling methods known in the art. Figures 26 and 27 show the
sequences, host string content, and structures of the C225 VL and VH domains. A GDR
graft of this antibody was constructed by placing the C225 CDRs into the context of the
frameworks of the most homologous human germlines, determined to be vlk_6D-21 for VL
and vh_4-30-4 for VH using the sequence alignment program BLAST. The sequences and
string content of these CDR grafts are shown in Figures 28 and 29, along with structures of
modeled C225 highlighting the mutational differences between the CDR grafted C225
variable chains and WT.
[192] Variants with reduced immunogenicity were generated by applying a string
optimization algorithm on the WT C225 VL and VH sequences, similar to as described above
for AC 10 except that single instead of multiple amino acid substitutions were sampled. HSC
of each sequence was optimized using a window size w=9, and the same set of CDR and
VLA/H interface proximal residues were masked. The calculation was run for C225 VL and
VH in 1 00 separate iterations, generating a set of diverse C225 variants with more host
string content than WT. Figure 30 shows the nonredundant set of output sequences from
these calculations for the C225 VL and VH regions, referred to as C225 VL HSC Calculation
1 and C225 VH HSC Calculation 1, respectively. In addition to the HSC score, the structural
consensus and structural precedence of each sequence was evaluated (USSN 60/528,229,
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WO 2005/056759
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filed December 8, 2003, entitled Protein Engineering with Analogous Contact Environments)
in order to evaluate its structural integrity.
[193] A second set of similar calculations were run on the C225 VL and VH sequences,
except that the algorithm was allowed to sample multiple amino acid substitutions, rather
than only single substitutions, in order to optimize HSC. Figure 31 shows the nonredundant
set of output sequences from these calculations for the C225 VL and VH regions, referred to
as C225 VL HSC Calculation 2 and C225 VH HSC Calculation 2, respectively. Here, two
measure of structural fitness, referred to as "Structural Consensus" and "Structural
Precedence" (USSN 60/528,229 and USSN 60/602,566), are used to evaluate the structural
and functional integrity of the sequences, in addition to HSC score. The output sequences
were clustered based on their mutational distance from the other sequences in the set, and
these clusters are delineated by the horizontal black lines in the Figure.
[194] The calculations described above and presented in Figures 30 and 31 were used to
generate a set of C225 VL and VH variants. In some cases, further substitutions were made
to output sequences, using string and structural scores, as well as visual inspection of the
modeled C225 structure, to evaluate fitness. Figures 32 - 40 present the sequences,
structural scores, string scores, and mapped mutational differences on the modeled C225
structure for each of the C225 VL and VH variants. Iteration 21 from C225 VL HSC
calculation 1 served as the precursor for L2 C225 VL, iteration 17 from C225 VL HSC
calculation 2 served as the precursor for L3 C225 VL, and iteration 38 from C225 VL HSC
calculation 2 served as the precursor for L4 C225 VL. Iteration 23 from C225 VH HSC
calculation 1 served as the precursor for H3, H4, and H5 C225 VH, iteration 5 from C225 VH
HSC calculation 2 served as the precursor for H6 C225 VH, iteration 41 from C225 VH HSC
calculation 2 served as the precursor for H7 C225 VH, and iteration 44 from C225 VH HSC
calculation 2 served as the precursor for H8 C225 VH.
[195] Tables 7 and 8 present the mutational, structural fitness, and host string content
scores for the C225 VL and VH variants as compared to the WT and CDR grafted C225
sequences. In addition, the maximum identity match to the germline for each string in the
sequences was also determined, referred to as N !D max. This represents the total number of
strings in each sequence whose maximum identity to the corresponding strings in the human
germline is the indicated value. For w = 9, Tables 7 and 8 list N 9 max, N 8 max, N 7 max, and
N^max for each sequence. Also provided is the framework region homogeneity. In addition
to the aforementioned structural and host string analysis, each sequence was analyzed for
its global homology to the human germline; tables 7 and 8 present the most homologous
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human germline sequence for each sequence (Closest Germline) and corresponding identity
to that germline (ID to Closest Germline), determined using the sequence alignment program
BLAST.
Table 7. C225 VL Variants
WT
CDR
Graft
L2
L3
L4
Mutations
25
17
21
18
Structural
Consensus
0.49
0.52
0.56
0.58
0.54
Oil UUIUI Cll
0.53
0.56
0.57
Precedence
0.59
0.57
ri uiTiai 1 o in rig
Content
0.79
0.94
0.91
0.92
0.91
Human String
Similarity
0.15
0.65
0.51
0.58
0.57
Framework Region
Homogeneity
0.97
0.52
0.50
0.78
N 9 max
13
69
52
60
58
N 8 max
27
15
28
24
23
N 7 max
37
22
20
16
19
N«&max
30
1
7
7
7
Closest
Germline
6D-21
6D-21
3-11
1D-13
6D-21
ID to Closest
63/95
87/95
72/95
73/94
79/95
Germline
66%
91%
75%
77%
83%
Table 8. C225 VH Variants
WT
CDR
Graft
H3
H4
H5
H6
H7
H8
Mutations
33
18
21
15
21
22
28
Structural
0.44
0.48
0.51
Consensus
0.46
0.49
0.52
0.49
0.53
Structural
Precedence
0.55
0.54
0.55
0.54
0.51
0.55
0.58
0.55
Human String
Content
0.67
0.84
0.79
0.81
0.77
0.79
0.79
0.79
Human String
Similarity
0.04
0.56
0.36
0.41
0.33
0.36
0.35
0.33
Framework Region
Homogeneity
0.97
0.45
0.52
0.50
0.50
0.76
0.77
N 9 max
3
66
42
48
38
42
41
39
N 8 max
23
17
25
24
17
24
21
27
N 7 max
32
10
19
16
26
23
25
23
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H8
30
3-33
ID to Closest 56 / 99 88 / 99 67 / 99 74 / 99 64 / 99 69 / 99 67 / 99 80 / 99
Germline 56% 88% 66% 74% 64% 70% 67% 81%
[196] Again, whereas the CDR grafted C225 antibodies are most homologous to a single
human germline sequence, the C225 variants of the present invention are homologous to
different human germline sequences in different regions of the sequence. Whereas CDR
grafted C225 VH is most homologous to human germline subfamily 4 across its entire
sequence, H4 C225 VH is most homologous to subfamily 4 in FR1 , subfamily 3 in FR2, and
subfamily 2 in FR3. Additionally, whereas the CDR grafted antibodies are most homologous
to a single germline sequence that is also the most homologous sequence to the parent
sequence, the present invention presents a set of antibodies for a given antibody that are
most homologous to different human germline sequences, which need not be the most
homologous germline sequence to WT. For example, Table 7 shows that CDR grafted C225
VL is most homologous to vIk_6D-21 , which is also the most homologous human germline to
WT C225. However L2, L3, and L4 are most homologous to three different human
germlines - vlk>1 1, vlk_1D-13, and vlk_6D~21 respectively. Thus the variants of the
present invention explore a substantially greater amount of diversity than CDR grafted
antibodies.
[197] The genes for the C225 variable regions were constructed as described above, and
subcloned into a modified pASK84 vector (Skerra, 1994, Gene 141: 79-84) comprising
mouse constant regions for expression as Fabs. Select C225 variants were experimentally
tested for their capacity to bind EGFR antigen. L2/H3 and L2/H4 C225 Fabs were
expressed from the pASK84 vector in E. Co// with a His-tag, and purified using Nickel-affinity
chromatography. Antigen affinity of the C225 variants was tested using SPR similar to as
described above. EGFR extracellular domain (purchased commercially from R&D Systems)
was covalently coupled to the dextrane matrix of a CM5 chip using NHS-linkage chemistry.
C225 Fabs were reacted with the EGFR sensor chip surface at varying concentrations.
Global Langmuir fits were been carried out for the concentrations series using the
BiaEvaluation curve fitting software. The on-rate constant (ka), off-rate constant (kd),
equilibrium binding constant (KD=kd/ka), and predicted saturation binding signal (Rmax)
derived from these fits are presented in Table 9, along with the Chi2 which quantifies the
WT CDR H3
Wl Graft
N^max 61 26 33
2EZ. «" "- 30 - 4 4 - M -
H4
H5
H6
H7
31
38
30
32
2-26
4-30-4
3-33
4-30-4
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average deviation of the fit curve from the actual data curve. The data indicate that both the
L2/H3 and L2/H4 C225 variants bind EGFR antigen.
Table 9. SPR data on C225 Variants
C225
ka (1/Ms)
kd (1/s)
KD(M)
Rmax
(RU)
Chi2
L2/H3
2.79 x10 4
5.35 X10 -3
1.92x1 0" 7
174
8.83
L2/H4
1.79 x10 4
4.73 X10- 3
2.64 x It)" 7
153
2.69
[198] In order to investigate the anti-EGFR variants in the context of a full length antibody,
the C225 WT (LO ad HO) and variant (L2, L3, L4, H3, H4, H5, H6 ? H7, and H8) regions were
subcloned into the mammalian expression vector pcDNA3.1Zeo (Invitrogen) as described
above. All combinations of the light and heavy chain plasmids Were co-transfected into 293T
cells, and antibodies were expressed, harvested, and purified as described above. Binding
of the C225 WT (L0/HO) and variant (L0/H3, L0/H4, L0/H5, L0/H6, L0/H7, L0/H8, L2/H3,
L2/H4, L2/H5, L2/H6, L2/H7, L2/H8, L3/H3, L3/H4, L3/H5, L3/H6, L3/H7, L3/H8, L4/H3,
L4/H4, L4/H5, L4/H6, L4/H7, and L4/H8) antibodies was determined using SPR similar to as
described above. Full length antibodies were flowed over the EGFR sensor chip described
above. Figure 41 shows the SPR sensorgrams obtained from the experiments. The curves
consist of a association phase and dissociation phase, the separation being marked by a
little spike on each curve. As a very rough approximation the signal level reached near the
end of the association phase can be used as an indicator for relative binding. For all the
curves this signal level is within 25% of the average level indicating that none of the antibody
variants have significantly lost their ability to bind to EGFR.
[199] To assess the capacity of the anti-EGFR antibodies to mediate effector function
against EGFR expressing cells, the C225 variants were tested in a cell-based ADCC assay.
Human peripheral blood monocytes (PBMCs) were used as effector cells, A431 epidermoid
carcinoma cells were used as target cells, and lysis was monitored by measuring LDH
activity using the Cytotoxicity Detection Kit as described above. Figure 42 shows the dose
dependence of ADCG at various antibody concentrations for WT and variant C225
antibodies. The results show that a number of the C225 variants have comparable or better
ADCC than WT C225 with respect to potency and efficacy. These data may be weighed
together with the antigen affinity data and other data to choose the optimal anti-EGFR
clinical candidate. As exemplified above with AC10 variants, combinations of the C225
variants of the present invention with amino acid modifications that alter effector function are
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contemplated.
Example 3. Immunogenicity Reduction of ICR62
[200] To further illustrate application of the method described in the present invention, and
to validate its broad applicability to immunogenicity reduction of proteins, an example is
provided using as the parent sequence the anti-EGFR antibody ICR62 (Institute of Cancer
Research) (PCT WO 95/20045; Modjtahedi et al., 1993, J. Cell Biophys. 1993, 22(1-3):129-
46; Modjtahedi et al., 1993, Br J Cancer. 1993, 67(2):247-53; Modjtahedi et al, 1996, Br J
Cancer, 73(2):228-35; Modjtahedi et al, 2003, Int J Cancer, 105(2):273-80). A structural
model of the rat ICR62 variable region was constructed using standard antibody modeling
methods known in the art. Figures 43 and 44 show the sequences, host string content, and
structures of the ICR62 VL and VH domains. A CDR graft of this antibody was constructed
by placing the ICR62 CDRs into the context of the frameworks of the most homologous
human germlines, determined to be vlk_1-17 for VL and vh_1-f for VH using the sequence
alignment program BLAST. The sequences and string content of these CDR grafts are
shown in Figures 45 and 46, along with structures of modeled ICR62 highlighting the
mutational differences between the CDR grafted ICR62 variable chains and WT.
[201] Variants with reduced immunogenicity were generated by applying a string
optimization algorithm on the WT ICR62 VL and VH sequences, similar to as described
above for AC10 except that single instead of multiple amino acid substitutions were
sampled. HSC of each sequence was optimized using a window size w=9, and the same
set of CDR and VLA/H interface proximal residues were masked. The calculation was run
for ICR62 VL and VH in 100 separate interactions, generating a set of diverse ICR62
variants with more host string content than WT. Figure 47 shows the nonredundant set of
output sequences from these calculations for the ICR62 VL and VH regions, referred to as
ICR62 VL HSC Calculation 1 and ICR62 VH HSC Calculation 1 respectively. In addition to
the HSC score, the structural consensus and structural precedence of each sequence was
evaluated (USSN 60/528,229, filed December 8, 2003, entitled Protein Engineering with
Analogous Contact Environments) in order to evaluate its structural integrity.
[202] The calculations described above and presented in Figure 47 were used to generate
a set of ICR62 VL and VH variants. In some cases, further substitutions were made to
output sequences, using HSC and Structural Precedence scores, as well as visual
inspection of the modeled ICR62 structure, to evaluate fitness. Figures 48 - 50 present the
sequences, host string content, and mapped mutational differences on the modeled ICR62
structure for each of the ICR62 VL and VH variants. Iteration 20 from ICR62 VL HSC
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calculation 1 served as the precursor for L2 ICR62 VL Iteration 1 from ICR62 VH HSC
calculation 1 served as the precursor for H9, and iteration 5 from ICR62 VH HSC calculation
2 served as the precursor for H10 ICR62 VH.
[203] Tables 10 and 11 present the mutational, structural fitness, and host string content
scores for the ICR62 VL and VH variants as compared to the WT and CDR grafted ICR62
sequences. In addition, the maximum identity match to the germline for each string in the
sequences, referred to as N (D max, is also provided, as well as the framework region
homogeneity. In addition to the aforementioned structural and host string analysis, each
sequence was analyzed for its global homology to the host germline; tables 10 and 1 1
present the most homologous host germline sequence for each sequence (Closest
Germline) and corresponding identity to that germline (ID to Closest Germline), determined
using the sequence alignment program BLAST,
Table 10. ICR62 VL Variants
WT
CDR
Graft
L2
Mutations
0
11
6
Structural
Consensus
0.56
0.60
0.61
Structural
Precedence
0.52
0.58
0.57
Human String
Content
0.86
0.91
0.90
Human String
Similarity
0.38
0.58
0.56
Framework Region
Homogeneity
0.62
0.97
0.64
N 9 max
37
59
56
N B max
26
21
19
N 7 max
31
18
22
N^max
13
9
10
Closest
Germline
1-17
1-17
1-17
ID to Closest
76/95 86/95 81 /9(
Germline
80%
90%
85%
Table 11. ICR62 VH Variants
WT CDR Graft H9 H10
Mutations 0 34 20 21
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WT
CDR Graft
H9
H10
otructurai
Consensus
0.43
0.44
0.46
0.45
Structural
rreceuence
0.42
0.52
0.47
0.49
Human String
oonient
0.64
0.85
0.79
0.79
Human String
Similarity
0.01
0.54
0.28
0.33
Framework Region
Homogeneity
1.00
0.64
0.85
N 9 max
1
64
33
39
N 8 max
16
24
33
30
N 7 max
35
14
28
25
N^max
67
17
25
25
Closest
Germline
1-f
1-f
1-f
1-f
ID to Closest
60/98
92/98
72/98
77/98
Germline
61%
93%
73%
79%
[204] Again, as observed from the significant differences in FRH and closest germlines, the
ICR62 variants are homologous to different host germline sequences in different regions of
the sequence. The genes for the ICR62 WT and L2/H9 variable regions were constructed
as described above, and subcloned into a modified pASK84 vector (Skerra, 1994, Gene 141:
79-84). The ICR62 Fabs experimentally tested for their capacity to bind EGFR antigen. WT
and L2/H9 ICR62 Fabs were expressed from the pASK84 vector in E. Coli with a His-tag,
and purified using Nickel-affinity chromatography. Antigen affinity of the ICR62 antibodies
was tested using SPR similar to as described above, with EGFR covalently coupled to the
CMS chip reacted with IGR62 antibodies at varying concentrations. The fits to the data, as
described above, are provided in Table 12. fits were been carried out for the concentrations
series using the BiaEvaluation curve fitting software. As can be seen, L2/H9 ICR62 binds
with comparable affinity as WT to the EGFR antigen.
Table 12. SPR data on ICR62 Variants
ICR62
ka
(1/Ms)
kd (1/s)
KD (M)
Rmax
(RU)
Chi2
WT
9.86 x
10 4
2.53x10"
5
2.57 x 10"
10
402
1.86
L2/H9
2.35 x
10 s
1.06x10-
4
4.50 x 10-
10
508
4.91
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Example 4. String diversity exploration of immunoglobulins
PCT/US2004/040694
[205] The generation of mutational diversity based on HSC is much broader than the
primary variant - secondary variant strategy described above for H3/L3 AC 10. Indeed
substitutions can be designed for any parent protein wherein the substitutions result in
positive or neutral impact on the host string content of the parent sequence. Again, the
advantage of such a strategy is that it generates a diverse set of minimally immunogenic
variants that have the potential for optimized properties, including but not limited to antigen
affinity, activity, specificity, solubility, expression level, and effector function. Such a set of
variants may be designed, for example, to explore diversity for other parent
immunoglobulins, including but not limited to nonhuman antibodies, humanized or otherwise
engineered antibodies (Clark, 2000, Immunol Today 21 :397-402), (Tsurushita & Vasquez,
2004, Humanization of Monoclonal Antibodies, Molecular Biology of B Cells, 533-545,
Elsevier Science (USA)), and "fully human" antibodies, obtained for example using
transgenic mice (Bruggemann ef a/., 1 997, Curr Opin Biotechnol 8:455-458) or human
antibody libraries coupled with selection methods (Griffiths ef a/., 1998, Curr Opin Biotechnol
9:102-108).
Example 5. Unique properties of variant proteins generated by the methods of the present
invention
[206] The methods described in the present invention generate variant proteins that
possess a number of unique properties relative to variant proteins generated by other
methods that attempt to achieve the same or similar goal. Figures 51 and 52 provide the
host string content (HSC, Equation 3), exact string content (ESC, Equation 3a), and
framework region homogeneity (FRH, Equation 10) of the AC10, C225, and ICR62 VH
(Figure 51 ) and VL (Figure 52) variants of the present invention, compared with a number of
antibody variable regions "humanized" by methods in the prior art. If a variant sequence's
exact string content is derived solely from a single germline sequence, the FRH would be
close to 1.0. Alternatively, as is the case with many of the variant sequences created by the
present invention, FRH values can be significantly less than 1, with values ranging from 0.4
to 1.0, indicating, as expected, that sequences with high exact string content can be
discovered with contributions from multiple germline subfamilies and sequences. At the
same time, the variant sequences engineered using the present invention have high host
string content, and thus are predicted to have low potential for immunogenicity in humans.
For example, as shown in Figure 51 variant VH sequences generated using the present
invention have HSC values generally higher than 75%, and many of them have FRH values
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WO 2005/056759
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lower than 0.6, indicating their HSC is derived from multiple germline frameworks. As shown
in Figure 52, similar trends apply for variant VL sequences generated using the present
invention
[207] Whereas particular embodiments of the invention have been described above for
purposes of illustration, it will be appreciated by those skilled in the art that numerous
variations of the details may be made without departing from the invention as described in
the appended claims. All references are herein expressly incorporated by reference.
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CLAIMS
We claim:
1 . A method of generating a variant protein for a host as compared to a parent protein,
comprising:
A. comparing said parent protein sequence with two or more natural protein
sequences from said host species;
B. analyzing one or more amino acid strings of said parent protein sequence
with a corresponding amino acid string of each natural protein sequence; and,
C. substituting one or more amino acids of said parent protein sequence with a
corresponding amino acid string of a natural protein sequence on an amino acid
string by amino acid string basis.
D. wherein said variant protein has increased host string content as compared to
said parent protein, and,
E. wherein said substituted amino acids include a first substitution in a first string
from a first natural protein, and a second substitution in a second string from a
second natural protein.
2. The method according to claim 1, wherein said substituted amino acid is a
consensus of two or more natural proteins.
3. The method according to claim 1, wherein said host species is human.
4. The method according to claim 1, wherein said parent protein family is murine.
5. The method according claim 1, wherein said natural protein sequences are germline
sequences.
6. The method according to claim 1, wherein one of the substitutions is not the
corresponding residue from the most homologous natural sequence.
7. The method according to claim 5, wherein one of the substitutions is not the
corresponding residue from a consensus of homologous natural sequences.
8. The method according to claim 1, wherein one of the substitutions is made at a
position that is not surface exposed.
9. The method according to claim 1, wherein said variant protein is an antibody.
10. The method according to claim 9, wherein one of the substitutions is not in a CDR.
70
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1 1 . The method according to claim 9, wherein one of the substitutions is made at a
position that is part of the VH/VL interface.
12. The method according to claim 9, wherein said variant antibody comprises an affinity
for an antigen no lower than about two-fold less than said parent protein.
13. The method according to claim 9, wherein said variant antibody comprises an affinity
for an antigen of about two-fold more than said parent protein.
14. The method according to claim 9, whereiq said variant protein comprises a
framework region identity as compared to a natural protein sequence of less than about
85%.
15. The method according to claim 9, wherein said variant protein comprises a
framework region homogeneity of less than about 0.6.
16. The method according to claim 9, wherein said variant antibody comprises no
substitutions within about 5 Angstroms of a CDR residue.
17. The method according to claim 9, wherein said antibody has modulated effector
function as compared to said parent antibody.
18. The method according to claim 17, wherein said effector function is ADCC.
19. The method according to claim 1, wherein more than one variant protein is generated
from said method to form a variant protein set.
20. The method according to claim 19, wherein said variant protein set comprises at
least two variant proteins that differ by more than about 5 amino acids.
21 . The method according to claim 19, further synthesizing at least one variant protein.
22. The method according to claim 1, wherein said analyzing one or more amino acid
strings is not applied to a CDR region.
23. The method according to claim 1, wherein said variant protein has at least one
improved property is selected from a group consisting of solubility, stability, expression,
affinity, immunogenicity, activity and functionality.
24. The method according to claim 1, wherein said analyzing one or more amino acid
strings includes theoretical binding to MHC agretopes.
25. The method according to claim 24, wherein said string is nine amino acids in length.
26. A computer readable memory to direct a computer to function in a specified manner
comprising:
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WO 2005/056759
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A. a comparison module to compare a parent protein sequence and two or more
host natural protein sequences;
B. an analyzing module to provide a comparison between a parent protein
sequence and one or more natural protein sequences;
C. a substitution module to replace one or more amino acids said parent protein
sequence with a corresponding amino acid string of a natural protein sequence on an
amino acid by amino acid basis,
D. wherein said variant protein has increased host string content as compared to
said parent protein.
27. A method of generating a variant protein for a host as compared to a parent protein,
comprising:
A. comparing said parent protein sequence with two or more natural protein
sequences;
B. substituting amino acids in said parent protein sequence with amino acids in
one or more natural protein sequences, wherein a substitution increases the
sequence identity of said variant protein to a natural protein sequence as compared
to said parent protein within a string,
C. wherein said variant protein has increased host string content as compared to
said parent protein.
28. A method of generating a variant protein for a host as compared to a parent protein,
comprising:
A. comparing said parent protein sequence with two or more natural protein
sequences from said host species;
B. analyzing one or more amino acid strings of said parent protein sequence
with a corresponding amino acid string of each natural protein sequence; and,
C. substituting one or more amino acids of said parent protein sequence with a
corresponding amino acid string of a natural protein sequence on an amino acid
string by amino acid string basis,
D. wherein said variant protein has increased host string content.
29. The method according to claim 28, wherein said substituted amino acid is a
consensus of two or more natural proteins.
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WO 2005/056759
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30. The method according to claim 28, wherein said host species is human.
31. The method according to claim 28, wherein said parent protein is murine.
32. The method according to claim 28, wherein said natural protein sequences are
germline sequences.
33. The method according to claim 28, wherein one of the substitutions is not the
corresponding residue from the most homologous natural sequence.
34. The method according to claim 33, wherein one of the substitutions is not the
corresponding residue from a consensus of homologous natural sequences.
35. The method according to claim 28, wherein one of the substitutions is made at a
position that is not surface exposed.
36. The method according to claim 28, wherein said variant protein is an antibody.
37. The method according to claim 36, wherein one of the substitutions is not in a CDR.
38. The method according to claim 36, wherein one of the substitutions is made at a
position that is part of the VH/VL interface.
39. The method according to claim 36, wherein said variant antibody comprises an
affinity for an antigen no lower than about two-fold less than said parent protein.
40. The method according to claim 36, wherein said variant antibody comprises an
affinity for an antigen of about two-fold more than said parent protein.
41. The method according to claim 36, wherein said variant protein comprises a
framework region identity as compared to a natural protein sequence of less than about
85%.
42. The method according to claim 36, wherein said variant protein comprises a
framework region homogeneity of less than about 0.6.
43. The method according to claim 36, wherein said variant antibody comprises less than
about 28 amino acid substitutions, as compared to a parent protein.
44. The method according to claim 36, wherein said variant antibody comprises no
substitutions within about 5 Angstroms of a CDR residue.
45. . The method according to claim 36, wherein said antibody has modulated effector
function as compared to said parent antibody.
46. The method according to claim 45, wherein said effector function is ADCC.
73
WO 2005/056759 PCT7US2004/040694
47. The method according to claim 28, wherein more than one variant protein is
generated from said method to form a variant protein set
48. The method according to claim 47, wherein said variant protein set comprises at
least two variant proteins that differ by more than about 5 amino acids.
49. The method according to claim 47, further synthesizing at least one variant protein.
50. The method according to claim 28, wherein said strings are not applied to a CDR
region.
51. The method according to claim 28, wherein said variant protein has at least one
improved property is selected from a group consisting of solubility, stability, expression,
affinity, immunogenicity, activity and functionality.
52. The method according to claim 28, wherein said analyzing one or more amino acid
strings includes theoretical binding to MHC agretopes.
53. The method according to claim 52, wherein said string is nine amino acids in length.
74
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1/61
PCT/US2004/040694
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QQQOQOQO
1 i
m i n vd r* to r- m (\ n
eo .NH_H«>^l^H^n_^r.^nn..
Lnvocoic.Htr.tr.ir.tr.r.iroir.1 i i
I I IQ> I Q I Q I Q l Q l > D i Q « Q Q Q
si. . I. s* ^ -------- - - -
II
, co cn | o o
-» c. oor.r.-.onO-'
oj « r. i i r. n i kj> \
»Q » Q Q i » Q i P
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III I
WO 2005/056759
4/61
PCT/US2004/040694
/
a -9
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CM
8
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h H H U) H O N
O I H I H I CN I
t a » Q i Q t Q
n rt o o o f*) n ro
WO 2005/056759 PCTYUS2 004/040694
5/61
Figure 1c
9 10
Rabat 678901234567
IGKJ1 WTFGQGTKVEIR
IGKJ2 YTFGQGTKLE I K
IGKJ3 FTFGPGTKVDIR
IGKJ4 LTFGGGTKVE IK
IGKJ5 I TFGQGTRLE I K
10 11
Rabat 1234567890123
IGHJ1 AEYFQHWGQGTLVTVSS
IGHJ2 YWYFDLWGRGTLVTVSS
IGHJ3 AFDVWGQGTMVTVSS
IGHJ4 YFDYWGQGTLVTVSS
IGHJ5 NWFDSWGQGTLVTVSS
IGHJ6 YYYYYGMD WGQGTTVTVS S
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Figure 2a
QVQLQQSGPE LVK PGASLKLSCT ASGFNI K
QVQLVQS GAE VKK P GASVKVSCK ASGYT F T
IDstringo=i5) = 6
Figure 2b
VH_m4D5 QVQLQQSGPELVK PGASLKLSCT ASGFNI K
VH_l-2 QVQLVQS GAE VKKPGASVKVSCKASGYTFT
VH_2-5 QITLKESGPTLVKETQTLTLTCTFSGFSLS
VH_3~ 7 EVQLVE S GGGLVQ PGGSLRLSCAASGFT F S
VH_4- 4 QVQLQESGPGLVKPPGTLSLTCAVSGGSI S
VH_5- 51 EVQLVQS GAE VKKPGE SLKISCKGSGYS F T
VH_6- 1 QVQLQQSGPGLVKPSQTLSLTCAI SGDSVS
VH_7- 4- 1 QVQLVQSGSELKKPGASVKVSCKASGYTFT
IDepltopeo=i5> = 6
IDepltopeo-nD = 4
IDepltopeo«i5) = 6
IDepltopeo=i5) = 3
IDepitbpecNis) = 6
IDepitopeo»i5) = 3
IDepltopeo=i5) = 6
v v ^
IDmaxo=i5) = 6
Figure 2c
VH_m4D5 QVQLQQSGPE LVK PGASLKLSCTASGFN IK
VH_l-2 QVQLVQS GAE VKK PGASVKVS CKASGYT F T
VH_2- 5 QI TLK ESGPT LVK PTQTLTLTCTF SGF SLS
VH_3- 7 EVQLVE S GGGLVQ PGG SLRLS CAASGFT F S
VH_4- 4 QVQLQESGPGLVKPPGTLSLTCAVSGGSIS
VH_5-51 EVQLVQSGAEVKKPGESLKI SCKGSGYSFT
VH_6-1 QVQLQQS GPGLVK PS QTL SLT CAI SGDSVS
VHJ7- 4- 1 QVQLVQS GSE LKK PGASVKV S CKASGYT F T
IDo=i)max = 9 IDo=i2)max = 6
IDo-2)max = 8 IDo=i3)max = 7
IDo=3)max = 8 |Do-i4)max = 7
IDo=^max = 8 lDo=i6)max = 6
IDcr=5)max = 8 IDo=ie)max = 6
lD(R8>max ~ 8 IDo=i7)max = 7
. IDo=7ynax = 7 IDo=»i8)max = 7
IE3(i=8)max - 7 IDo=i9)max = 7
IDo=e)max = 7 IDo=20)max = 7
ID(Rio)max = 7 IDo»2i)max = 6
IDo=n)max = 7 lDo=*22)max = 5
HSC(s) = 78.3
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Figured L0AC10VL
Figure 3a
DIVLTQSPASLAVSLGQRATISCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESG
IPARFSGSGSGTDFTLNIHPVEEEDAATYYCQQSNEDPWTFGGGTKLEIK
Figure 3b
FR1 FR2 FR3 FR4
Figure 3c
FR1 FR2 FR3 FR4
una
ux p.
HTii !
T
t.
fiJ
■ ASzr:.-..t-~.
"i'it
: i
1
■*■
T
.CI
......
:;uiixff
tr j
1H
— 1 — mm
* r Hff
t .
■""til ' !:
p-lifTi
:;Q
4 1 -4
* - p ■:- i
i 1; >&
; • •; I
;
1
n
il
t •* nH
I SH£
&.4.i!--.;|y--
iri"
.:"4- l . j.
i: ■■:]:: . >:.i
: |= !••
it r ^""
rr-t
i r * i
: ! - •
r.i_ -r
Figure 3d
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Figure 4. H0AC10VH
Figure 4a
QIQLQQSGPEWKPGASVKISCKASGYTFTDYYITWVKQKPGQGLEWIGWIYPGSGNTKYN
EKFKGKATLWDTSSSTAFMQLSSLTSEDTAVYFCANYGNYWFAYWGQGTQVTVSA
Figure 4b
Figure 4c
FR1 FR2 FR3 FR4
i !..L.L.g =
eli.
- ^
""1
■ !-•
-; j.! - . .;.;:_!■-
l.r-L
1 1
t
J
. :.]
• • -i
[ j i j i
in i ;!;
If
i
j'
1 : -
$
• L
i
1
i
f
T
-;:.tT|
i « I
Itl
-r t--i-!. %ri=Hi
*~ + » 1 "* J ■ iT
M
j_
■ ■. : i n rr i ;
i I
; > ; i 1 1 i t
~T~!
Figure 4d
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Figures. CDR grafted AC10 VL
Figure 5a
DIVMTQSPDSLAVSLGERATINCKSSQSVDFDGDSYLAWYQQKPGQPPKLLIYAASNLESG
VPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQSNEDPWTFGGGTKLEIK
FR1
Figure 5b
FR2
FR3
FR4
Figure 5c
FR1 FR2 FR3 FR4
Figure 5d
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Figure 6. CDR grafted AC10VH
Figure 6a
QVQLVQSGAEVKKPGASVKVSCKASGYTFTDYYMHWVRQAPGQRLEWMGWIYPGSGNT
KYSQKFQGR\n"ITRDTSASTAYMELSSLRSEDTAVYYCARYGNYWFAYWGQGTLVTVSS
Figure 6b
Figure 6d
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Figure 8. L1 AC10VL
Figure 8a
DIVLTQSPATLSLSPGERATLSCRASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLES
GIPARFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPWTFGGGTKVEIK
FR1
Figure 8b
FR2
FR3
S IIM
FR4
FR1
Figure 8c
FR2
FR3
FR4
rT • ii'TiriM— i •! r~'
r^ft ■
e
p — [
:'C:|.
!
, "i
Li. MJ ..J..:..; i. ..
;-r|=n- itr -!-"-;
ti- !
i i
Mr- tip
- aa--!~
M _ -:
; K~ : -Vj- '{I
_L..t ~
Figure 8d
WO 2005/056759
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Figure 9. L2 AC10VL
Figure 9a
DIVLTQSPSSLSASVGDRVTITCRASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESG
IPARFSGSGSGTDFTLTISSLQPEDFATYYCQQSNEDPWTFGGGTKVEIK
Figure 9b
FR1 FR2 FR3 FR4
Figure 9d
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Figure 10. L3AC10VL
Figure 10a
DIVLTQSPDSLAVSLGERATINCKASQSVDFDGDSYMNWYQQKPGQPPKVLIYAASNLESG
IPARFSGSGSGTDFTLTINSLEAEDAATYYCQQSNEDPWTFGGGTKVEIK
Figure 10b
FR1 FR2 FR3 FR4
Figure 10d
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Figure 11. H1 AC10VH
Figure 11a
QIQLVQSGPEVKKPGASVKVSCKASGYTFTDYYITWVRQAPGQGLEWMGWIYPGSGNTK
YNEKFQGRVTITVDTSASTAYMELSSLRSEDTAVYFCANYGNYWFAYWGQGTLVTVSS
Figure 11b
Figure 11c
FR1 FR2 FR3 FR4
1 1
...UBS
sar
\
~J '
-U
„l..
— —
ti
hJ
iiiR
j.. j.
f If 4 :-H
. .. .
-"•-{
f-ix^3|Z^:. j-rr-j-- -ft:"
— ^
II
s
^■
— \ -r-
* t :-\ v: ; . r 4, - i - "7. i-
— t-i •* i
Figure 11d
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Figure 12. H2AC10VH
Figure 12a
QIQLVESGGGLVKPGGSLRLSCAASGYTFTDYYITWVRQAPGQGLEWMGWIYPGSGNTK
YNEKFQGRVTMTVDTSTSTAYMELSSLRSEDTAWFCANYGNYWFAYWGQGTLV7VSS
Figure 12b
FR1 FR2 FR3 FR4
Figure 12c
FR1 FR2 FR3 FR4
! i l! j i"
1 1 •
: 1
I • '
-
J '■
1 1.
r-^-fi:: r -^fi-
|
T..TZZ
:.- .
f sr -sr. \ :>-*•
? \ *
*■ f
" ""I
T"
-i • i i-i-: -i 1-+
-H
- i
Figure 12d
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Figure 13. H3AC10VH
Figure 13a
QIQLVQSGPEVKKPGASVKVSCKASGYTFTDYYITWVRQAPGQGLEWMGWIYPGSGNTK
YNEKFQGRFVFSVDTSASTAYLQISSLKAEDTAVYFCANYGNYWFAYWGQGTLVTVSS
FR1
FR2
Figure 13b
FR3
FR4
FR1
FR2
Figure 13c
FR3
FR4
i
r
• 4
'i —
j
!
r --. • r
!:~ !
M 11
1 i !; !
«
j.
f-
S.S
r;
i
... .....
J. .
V.
..!.....
■m
...!_. .:.:«..!
i :■ i- !
— 1 — 1
ill. i't:' ! .
i
til IS;
; h
1
i : i 1
! if!
J .
■ ii rr™'" t-
i i *
!■ . , 1
H ' • .
i
Figure 13d
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Figure 14a
100H
o
u
§ 80
(/)
0)
c
E
3
—I
E
s
5
60
40
20-
1 1 1 1 1 1 1 r 1 —
-11 -10 -9 -8 -7
log [antibody] (0.2M>
Figure 14b
iooH
o
u
§ 80
CO
<D
c
E
3
60
E 40
X
20-
-12
— i —
-11
i
-10
-9
— i—
-8
-7
— T—
-6
log [antibody] (0.2M)
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Figure 17a
O 1 2
log [antibody] (ng/ml>
□ HOLD
O H0L1
O H0L2
A H0L3
s H1L0
o H1L1
o H1L2
A H1L3
Figure 17b
0 1 2
tog [antibody] (ng/ml)
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Figure 18b
35-i
log [antibody] (ng/ml)
■ H0L0
• H3L3
Q H3L3, 1332E
H3L3, V264I/I332E
* H3L3, S239D
A H3L3, S239D/I332E
V H3L3
S239D/A330UI332E
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Figure 19
log [antibody] (0.1M)
i
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Figure 20. L3.71 AC10VL
Figure 20a
EIVLTQSPDSLAVSLGERATINCKASQSVDFDGDSYLNWYQQKPGQPPKVLIYAASTLQSG
VPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSNEDPWTFGGGTKVEIK
Figure 20b
FR1 FR2 FR3 FR4
Figure 20c
FR1 FR2 FR3 FR4
Figure 20d
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PCT/US2004/040694
Figure 21. L3.72AC10VL
Figure 21a
AIVLTQSPDSLAVSLGERATINCKASQSVDFDGDSYLNWYQQKPGQPPKVLIYAASTLETG
VPSRFSGS GSGTD FTLTI N SLEAE DAATYYCQQS NEDPWTFGGGTKVE IK
Figure 21b
FR1 FR2 FR3 FR4
Figure 21 d
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Figure 22. H3.68 AC10 VH
Figure 22a
QLQLVQSGPEVKKPGASVKVSCKVSGYTFTDYYITWVRQAPGQALEWMGWIYPGSGNTK
YNEKFQGRFVFSVDTSASTAYLQISSLKAEDTAVYFCANYGNYWFAYWGQGTLV7VSS
FR1
FR2
Figure 22b
FR3
FR4
FR1
FR2
Figure 22c
FR3
FR4
-T-T.-
-rr-r
fir
r---: — H--H-T-
TTT
■ I «
llTBP, j
1I_L
Figure 22d
WO 2005/056759 PCT/US2004/040694
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Figure 23. H3.69AC10VH
Figure 23a
QLQLVQSGAEVKKPGASVKVSCKVSGYTFTDYYITVVVRQAPGQALEWMGWIYPGSGNTK
YSQKFQGRFVFSVDTSASTAYLQISSLKAEDTAVYYCANYGNYWFAYWGQGTLVTVSS
Figure 23b
FR1 FR2 FR3 FR4
Figure 23c
FR1 FR2 FR3 FR4
._L_L__U: •. ! !
•4--:--
VT "
'■ \ '
_1lJ_:
!
— i-^- - -I !!"
T-; r ;]|!
; — 1
i i
L 1
m ■
! .. ! li.i
- .Hi
i { » "" " ; >l?i t 1m - , > ™ i i I > ■ "i ■ r " f ' 7~ » i I • ; »i ■
Figure 23d
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PCT7US2004/040694
Figure 24. H3.70AC10VH
Figure 24a
QLQLVQSGAEVKKPGASVKVSCKVSGYTFTSYYISWVRQAPGQALEWMGWIYAGSGNTK
YSQKFQGRFVFSVDTSASTAYLQISSLKAEDTAVYYCANYGNYWFAYWGQGTLVTVSS
Figure 24b
FR1 FR2 FR3 FR4
Figure 24c
FR1 FR2 FR3 FR4
BBC
yf---. :
: 5 . ! r.:
I . I L . .
iHI
)
t *
T Oi i
L ~ . ! - - . J_
i
■ J ;
rr---4
. ill ;. i
-r- >- -
-jr.- -;-.}•_■
|._Uu
- * •!
• J • |->rr;^.th_.:-: r\
•»-!•: •' ;- • |4'-
• 1 "
HI- p.- .:■
;» t
1 !
" 1 .
i i: ,
i
Figure 24d
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Figure 25. Optimized Anti-CD30 lgG1 Antibodies
Figure 25a. Anti-CD30 Light Chain
EIVLTQSPDSLAVSLGERATINCKASQSVDFDGDSYLNWYQQKPGQPPKVLIYAASTLQSG
VPSRFSGSGSGTDFTLTINSLEAEDAATYYCQQSNEDPWTFGGGTKVEIK RTVAAPSVFIFP
PSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTL
TLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC
Figure 25b. Anti-CD30 IgG1 Heavy Chain Comprising Possible Fc Variants
QLQLVQSGAEVKKPGASVKVSCKVSGYTFTSYYISWVRQAPGQALEWMGWIYAGSGNTK
YSQKFQGRFVFSVDTSASTAYLQISSLKAEDTAVYYCANYGNYWFAYWGQGTLVTVSSA S
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPX,VF
LFPPKPKDTLMISRTPEVTCWXzDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
WSVLTVLHQDWLNGKEYKCKVSNKALPX3PX4EKTISKAKGQPREPQVYTLPPSRDELTKN
QVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGN
VFSCSVMHEALHNHYTQKSLSLSPGK
Position
EU Index
Position
WT
Possible Substitutions
239
S
D, E, N, Q, T
x 2
264
V
I.T.Y
x 3
330
A
Y.L.I
X4
332
I
D, E, N, Q
Figure 25c. Anti-CO30 Fc Variant lgG1 Heavy Chain
QLQLVQSGAEVKKPGASVKVSCKVSGYTFTSYYISWVRQAPGQALEWMGWIYAGSGNTK
YSQKFQGRFVFSVDTSASTAYLQISSLKAEDTAVYYCANYGNYWFAYWGQGTLVTVSSA S
TKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKAEPKSCDKTHTCPPCPAPELLGGPDVF
LFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYR
WSVLTVLHQDWLNGKEYKCKVSNKALPLPEEKTISKAKGQPREPQVYTLPPSRDELTKNQ
VSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNV
FSCSVM HEALHNHYTQKSLSLSPGK
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Figure 25d. Anti-CD30 lgG2 Heavy Chain Comprising Possible Fc Variants
QLQLVQSGAEVKKPGASVKVSCKVSGYTFTSYYISWVRQAP6QALEWMGWIYAGS6NTK
YSQKFQGRFVFSVDTSASTAYLQISSLKAEDTAVYYCANYGNYWFAYWGQGTLVTVSSA S
TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVtVSWNSGALTSGVHTFPAVLQSSGLY
SLSS\A/TVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPZ 1 Z 2 Z3Z 4 GPX 1 VF
LFPPKPKDTLMISRTPEVTCWXzDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFR
WSVLTVVHQDWLNGKEYKCKVSNKZ5LPX3PX4EKTISKTKGQPREPQVYTLPPSREEMTK
NQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQG
NVFSCSVMHEALHNHYTQKSLSLSPGK
Position
EU Index
Position
WT
Possible Substitutions
239
S
D, E, N, Q, T
x 2
264
V
I, T, Y
x 3
330
A
Y, L,l
x 4
332
I
D, E, N, Q
Z1
233
P
E
z 2
234
V
L
z 3
235
A
L
Z4
236
G
z 5
327
G
A
Figure 25e. Anti-CD30 Fc Variant lgG2 Heavy Chain
QLQLVQSGAEVKKPGASVKVSCKVSGYTFTSYYISWVRQAPGQALEWMGWIYAGSGNTK
YSQKFQGRFVFSVDTSASTAYLQISSLKAEDTAVYYCANYGNYWFAYWGQGTLVTVSSA S
TKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLY
SLSSVVTVPSSNFGTQTYTCNVDHKPSNTKVDKTVERKCCVECPPCPAPELLGGPDVFLFP
PKPKDTLMISRTPEVTCVWDVSHEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTFRWS
VLTWHQDWLNGKEYKCKVSNKGLPLPEEKTISKTKGQPREPQVYTLPPSREEMTKNQVS
LTCLVKGFYPSDIAVEWESNGQPENNYKTTPPMLDSDGSFFLYSKLTVDKSRWQQGNVFS
CSVMHEALHNHYTQKSLSLSPGK
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Figure 26. C225WTVL
Figure 26a
DILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSG
SGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELK
Figure 26b
Figure 26c
FR1 FR2 FR3 FR4
?.•!». .i .... . if.....
> ' T" ~ V ~
in:"":
itr
u
y.
1-
1
~*"T' ~
TLTi:-
im.
! i I r
•LfLl
-Li L
liilllilHi !
■E'
"■wrap i - •
j_:
r -
i
" i t
?r
±,.. r r , t j
it i
t.-ti !l
; ! -4
tH|pa~"r.'
i .
i-
j I- • i
- •; f
s . 1
i '
r..~ i
h--
i
■n----
.L
i
J_
■ — i "
i.~
' "
""f r
- : • ■«-■-■■. — |>r .
-..-i - . .1-
! . i
Figure 26d
WO 2005/056759
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Figure 27. WTC225VH
Figure 27a
QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDY
NTrPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQ
Figure 27b
FR1
FR2
Figure 27c
FR3
FR4
ii i »
', ■"' I '
|: « !
• ; > ■ j. .
i ! 1
i
: : - | ; : ; s
- i; i
■ 1 ! i
-r
i . i
ill.:..
in
! i
r 1
1
i Mi i
• i-.;
--.i.ri
-t-1
. i
; | r
!>: s : I
i* •
1 !
f
' ' * It
!
1 1 « ' t
i
i t ! ■ . • , i ii • i : .
• j-
L -!—t
Figure 27d
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Figure 28. CDR grafted G225 VL
Figure 28a
EIVLTQSPDFQSVTPKEKVTITCRASQSIGTNLHWYQQKPDQSPKLLIKYASESISGVPSRF
SGSGSGTDFTLTINSLEAEDAATYYCHQNNNWPTTFGAGTKLEIK
FR1
Figure 28b
FR2
FR3
FR4
Figure 28d
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PCTAJS2004/040694
Figure 29. CDR grafted C225 VH
Figure 29a
QVQLQESGPGLVKPSQTLSLTCTVSGFSLTNYGWSWIRQPPGKGLEWIGYIWSGGNTYYN
PSLKSRVTISVDTSKNQFSLKLSSVTAADTAVYYCARALTYYDYEFAYWGQGTLVTVSS
FR1
Figure 29b
FR2 FR3
HS ||l|ip;
FR4
FR1
Figure 29c
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15.14 Confidentiality.
(a) No Limited Partner shall disclose to any Person any information related to the
General Partner, the Partnership, any Principal, any Parallel Investment Vehicle, any Alternative
Investment Vehicle, any Portfolio Company or proposed Portfolio Company or any of their
respective Affiliates, in each case, that is not publicly available (or that is publicly available as a
result of a disclosure by such Limited Partner or any director, employee, officer, agent, legal,
financial or tax advisor of such Limited Partner in violation of this Section 04444 ); provided,
however, that nothing contained herein shall prevent any Limited Partner from furnishing (i) any
| re quir e d information that such Limited Partner is required to provide to any governmental
regulatory agency, self-regulating body or in connection with any judicial, governmental or other
| regulatory proceeding or as otherwise required by any applicable law , rule or regulation
(provided that any disclosure that is either (x) not to a governmental regulatory agency or (y) not
on a confidential basis, shall require prior written notice thereof to the General Partner) or (ii)
any information, so long as such disclosure is for a bona fide business purpose relating to the
such Limited Partners investment in the Partnership of such Limit e d Partner , to directors,
officers, employees and legal, financial and tax advisors of such Limited Partner who are
informed of the confidential nature of the information and who agree to be bound by the
provisions of this Section 04444 or who are otherwise .bound by substantially similar obligations
of confidentiality, and each Limited Partner agrees to b e boun d h e r e bv j [fn^po^^uciii
feu^rgm^^ 1
limitation of the foregoing, each Limited Partner acknowledges that notices and reports to such
Limited Partner hereunder may contain material non-public information concerning, among
other things, Portfolio Companies, and agrees not to use such information other than in
connection with monitoring its investment in the Partnership and agrees, in that regard, not to
trade in publicly traded Securities on the basis of any such information. Furthermore, the
Partners hereby acknowledge that pursuant to § 17-305(f) of the Act, the rights of a Limited
Partner to obtain information from the Partnership shall be limited to only those rights provided
for in this Agreement, and that any other rights provided under § 17-305(a) of the Act shall not
be available to the Limited Partners or applicable to the Partnership.
(b) In order to preserve the confidentiality of certain information disseminated by the
General Partner or the Partnership under this Agreement that a FOIA Limited Partner [that i s
s ub ject' to FQIA o r any Li m i ted Partner that has "one or more equity own e rs that are s ubj e ct to
FyQlA (any such Lim i t e d Partn e r, a " FOIA Li mite d Partner ; )j is entitled to receive pursuant to the
provisions of this Agreement, including, without limitation, quarterly, annual and other reports
(other than Schedule K-ls), information provided to the Advisory Committee and any
information provided at meetings of the Limited Partners, the General Partner may (i) provide to
such FOIA Limited Partner access to such information only on the Partnership's (or
Management Company's) website in password protected, non-downloadable, non-printable
| format, er-(ii) require such FOIA Limited Partner to return any copies of information provided to
it by the General Partner or the Partnership (including any subsequent copies made by such
Limited Partner) to the extent not prohibited by applicable law or regulation or (iii) lifefl .
(c) Notwithstanding the provisions of Section 04444, the General Partner agrees that
each Limited Partner that (x) is a private fund of funds (or other similar private collective
investment vehicle) having reporting obligations to its investors and (y) has, prior to the date on
( Commen t [M2]: Move lo definitions
^omment j [M 3]: :'- add mutual "
which such Limited Partner was admitted to the Partnership, notified the General Partner in
| writing that it is {electing the benefits of, this Section 1.1 (c) 1 5 . 14 (c) may, in order to satisfy such
reporting obligations, provide the following information to its investors (but only to the extent
that such investors are informed of the confidential nature of the information and either agree to
| be bound by the provisions of this Section Q44t4-4 or are otherwise bound by substantially similar
obligations of confidentiality): (i) the name and address of the Partnership; (ii) the fact that such
Limited Partner is a limited partner of the Partnership and the Partnership's general investment
strategy; (iii) the identity of the Qeneral Partner and any Principal; (iv) the final closing date of
the Partnership; (v) the amount of such Limited Partner's Commitment; (vi) the total amount of
such Limited Partner's Capital Contributions; (vii) the total amount of distributions received by
such Limited Partner from the Partnership; (viii) such Limited Partner's net internal rate of return
with respect to the Partnership's performance as a whole as prepared by such Limited Partner;
| [(ix) the name of any Portfolio Company, a description of the business of such Portfolio
Company and information regarding the industry and geographic location of such Portfolio
| Company; and (x) the cost of the Partnership's investment in a Portfolio [Compariyjl. With
respect to any disclosure referred to in clauses (i) through (x) above, each Limited Partner shall
indicate that such disclosure was not prepared, reviewed or approved, by the General Partner or
the [Parthfeship].
(d) Each Limited Partner shall promptly notify the General Partner if at any time such
Limited Partner is or becomes subject to Section 552(a) of Title 5 of the United States Code
(commonly known as the "Freedom of Information Act") or any public disclosure law, rule or
| regulation of any governmental body or non-governmental regulatory entity that could require
similar or broader public disclosure of confidential information provided to such Limited Partner
(collectively such laws, rules or regulations, " FOIA "). To the extent that any such Limited
Partner receives a request for public disclosure of any confidential Partnership information
| provided to it, such Limited Partner agrees that: (i) it shall use its reasonable best efforts to (*w)
promptly notify the General Partner of such disclosure request and promptly provide the General
Partner with a copy of such disclosure request or a detailed summary of the information being
requested, (yx) inform the General Partner of the timing for responding to such disclosure
request, (zy) consult with the General Partner regarding the response to such disclosure request;
(hz) it sha ll us e comm e rcial l y rea s onabl e effort s to oppose and prevent the requested disclosure
unless (A) such Limited Partner is advis e d determines in good faith upon advice of by-counsel
that there exists no reasonable basis on which to oppose such disclosure or (B) such disclosure
| relates solely to the information contained in clauses (i) through fcviuijof Section I . I Cc ) 1 5.1 1(c)
(and does not include any information relating to individual Portfolio Companies and/or copies
of this Agreement or related documents); and (iii) notwithstanding any other provision of this
Agreement, the General Partner may, in order to prevent any such potential disclosure that the
General Partner determines in good faith is [likejy.tp q'ccurj, withhold all or any part of the
information otherwise to be provided to such Limited Partner; provided, however, that the
General Partner shall not withhold any such information if such Limited Partner confirms in
writing to the General Partner, based upon advice of counsel, that compliance with the
procedures in Section 1 . 1(b) l 5.1 Kb) is legally sufficient to prevent such potential disclosure.
Comment [M4]:;ShouId not that this
t:'eliectibn;be'1riclud^'ih''sub'scnption :
Comment [M5]: Expand list:
management fees, date of investment,
j public Hqmdity.eyents -
Comment [ M 16]: Add assurance that
Comment [M7]: Update if additional
items added but exclude any portfolio
company information if listed above
• ^Comment [M8]: Any lower standard?
(e) The obligations and undertakings of each Limited Partner under this Section
0 15.11 shall be continuing and shall survive termination of the Partnership and this Agreement.
Any restriction or obligation imposed on a Limited Partner pursuant to this Section 044rM may
be waived by the General Partner in its discretion. Any such waiver or modification by the
General Partner shall not constitute a breach of this Agreement or of any duty stated or implied
in law or in equity to any Limited Partner, regardless of whether different agreements are
reached with different Limited Partners.
(f) The parties hereto agree that irreparable damage would occur if the provisions of
this Section 04444 were |^^^^[.. It is accordingly agreed that the parties hereto shall be
entitled to see k an injunction or injunctions to prevent breaches of this Section 04444 and to
enforce specifically the terms and provisions hereof in any court of the U.S. or any state having
jurisdiction, in addition to any other remedy to which they are entitled at law or in equity.
(g) Add Tax Carve out
; also* agree, not to raise as , a defen sc -
^--- { Formatted: Bullets and Numberin g J
(h) Add Delaware 305(b) boilerplate about ability to withhold
EXHIBIT K
Pipeline and Utility Easements Deed - by Owners to Mineral Owners and
Mineral Lessee
KJbCUKlJllNU Kbl^Ubo 1 bU Jt5 Y .
Chicago Title Company
WHEN RECORDED MAIL TO:
Esperson/Grimm
c/o Kronick Moskovitz
Tiedemann & Girard
1675 Chester Avenue, Suite 320
Bakersfield, CA 93301
Attention: Teri A. Bjorn, Of Counsel.
The undersigned Grantors declare:
Documentary transfer tax is: $
( ) computed on full value of property conveyed, or
( ) computed on full value less value of liens and
encumbrances remaining at time of sale.
( ) Unincorporated area of County of Solano, or
() City of Rio Vista.
GRANT OF PIPELINE AND UTILITY EASEMENTS
THIS GRANT OF PIPELINE AND UTILITY EASEMENTS (this "Easement Deed") is
made and entered into as of , 200 (the "Effective Date") by and among:
(1) THE GRIMM-RIO VISTA FAMILY LIMITED PARTNERSHIP, a
California limited partnership; RICHARD W. ESPERSON, also known as RICHARD W.
ESPERSON, JR., and IRENE S. ESPERSON, TRUSTEES OF THE RICHARD W.
ESPERSON AND IRENE SUE ESPERSON FAMILY TRUST DATED OCTOBER 17,
1991; MARK ESPERSON; GARY ESPERSON; STEPHEN ESPERSON; and KIMBERLY
ESPERSON (collectively the "Surface Owners"), the fee owners of that certain real property
legally described in Exhibit A-l (the "Property").
- 1 -
(2) RIO VISTA HILLS HOLDING COMPANY, LLC, a Delaware limited
liability company ("RVHHC"), who has an option to purchase that portion of the Property
shown and identified on Exhibit B as the Sale Property (the "Sale Property"). The remainder of
the Property shown and identified on Exhibit B , other than the Sale Property, is referred to as the
"Retained Property/' Surface Owners and RVHHC are collectively sometimes referred to herein
as the "Grantors."
(3) NORMA JEAN GRIMM, also known as JEAN HARRIS GRIMM, AS
TRUSTEE OF THE TRIMM FAMILY TRUST DATED OCTOBER 4, 1990 -
SURVIVORS TRUST; RICHARD W. ESPERSON, JR.; JOAN ESPERSON WEDDELL;
DAVID SANTOS; RICHARD SANTOS; STEPHEN ESPERSON; GARY ESPERSON;
MARK ESPERSON; SUSAN A. BORGESEN, formerly SUSAN A. WOODWORTH;
SANDRA J. DICKSON, formerly SANDRA GRIMM; SHARON E. HARRIS, formerly
SHARON GRIMM; STEPHEN A. GRIMM, formerly STEPHEN GRIMM; THE GRIMM-
RIO VISTA FAMILY LIMITED PARTNERSHIP, a California limited partnership; JENA
HARRIS GRIMM; DAVID L. SANTOS, SURVIVING TRUSTEE OF THE DAVID L.
AND LAURA E. SANTOS REVOCABLE TRUST DATED FEBRUARY 12, 2002; and
RICHARD W. ESPERSON, also known as RICHARD W. ESPERSON, JR., and IRENE S.
ESPERSON, TRUSTEES OF THE RICHARD W. ESPERSON AND IRENE SUE
ESPERSON FAMILY TRUST DATED OCTOBER 17, 1991 (collectively the "Mineral
Owners," each being a "Mineral Owner"), the owners of the reversionary interest in that certain
mineral estate (the "Mineral Estate") in, on and under the Property and comprised of that portion
of Parcel One and all of Parcels Two, Three and Four shown within bold line on the diagram
attached as Exhibit C-l , which owners are listed as to each of such Parcels One, Two, Three and
Four in Exhibit C-2 . The Mineral Estate is legally described in Exhibit C-3 .
(4) The owner of the fee simple determinable interest in the Mineral Estate (by virtue
of the grant of oil and gas lease concerning the Mineral Estate), ROSETTA RESOURCES
OPERATING, L.P., a. Delaware limited partnership, successor to Calpine Natural Gas
California, Inc., and Sheridan California Energy, Inc., both California corporations (the "Mineral
Lessee"). For this Easement Deed, the exclusive fee simple determinable interest held by
Mineral Lessee may be referred to as a mineral leasehold interest. The mineral leasehold estate
of Mineral Lessee also includes rights in and to the real property located outside of the bold line
on Exhibit B-l (the "Adjacent Property") and rights in and to that mineral estate in, on and under
the Adjacent Property (the "Adjacent Mineral Estate"), which together with Mineral Lessee's
leasehold rights in and to the Property and the Mineral Estate is referred to collectively as the
"Leasehold Estate." The Leasehold Estate encumbers all of the Parcels shown on Exhibit C-l
and is legally described in Exhibit C-4 . Mineral Owners and Mineral Lessee are collectively
referred to herein sometimes as the "Grantees."
Surface Owners, RVHHC, Mineral Owners and Mineral Lessee are sometimes
collectively referred to herein as the "Parties" or individually as a "Party."
RECITALS:
A. Surface Owners own the Property, excepting the Mineral Estate, which is
comprised of approximately 504 assessed acres of undeveloped real property in the City of Rio
Vista ("City"), County of Solano, State of California, Assessor's Parcel Nos. 49-310-040, 49-
3 1 0-300, 49-3 1 0-020 and 49-3 10-010. A diagram of the Property is attached as Exhibit B .
B. Surface Owners have entered into an Option Agreement (the "Option Agreement")
with RVHHC to sell the Sale Property for development of a residential/commercial project,
including school sites, recreation/park sites, and open space areas and public uses (the "Project") as
generally shown on Exhibit D . The Retained Property shown on Exhibit B will be retained for
development by Surface Owners as" part of an overall master-planned community to be known as
Del Rio Hills, which is currently planned to contain only the Sale Property and the Retained
Property. This Easement Deed covers the Property which includes both the Sale Property and the
Retained Property.
C. As used in this Easement Deed, "Owner" means both Surface Owner and RVHHC
and their respective heirs, assigns, transferees and successors. However, for purposes of
apportioning responsibility and liability for the obligations generally ascribed to "Owner" in this
Easement Deed, and unless the context of this Easement Deed otherwise specifically provides, (i)
the fee estate owner of the Sale Property shall be responsible and liable only for those obligations,
actions and activities arising under this Easement Deed that pertain to or otherwise stem from the
ownership of the Sale Property and (ii) the fee estate owner of the Retained Property shall be
responsible and liable only for those obligations, actions and activities arising under this Easement
Deed that pertain to or otherwise stem from the ownership of the Retained Property. If the Option
Agreement terminates or is terminated prior to RVHHC acquiring any portion of the Property,
the term RVHHC shall mean Surface Owners or future developers taking their interest through
Surface Owners, and RVHHC shall not have any further rights under the Accommodation
Agreement (as defined in Recital G herein) or this Easement Deed.
D. All oil, natural gas, casinghead gas, condensate and other hydrocarbon substances in,
on and under the Property are part of the Mineral Estate.
E. Mineral Lessee is the successor lessee under an Oil and Gas Lease between
Edward Drouin, also known as E. D. Drouin, as lessor, and Amerada Petroleum Corporation of
California, as lessee, dated May 28, 1935 (the "Lease"), and recorded on October 9, 1935, in
Book J 51, Page 72, Official Records of Solano County, California. The Lease covers 912 acres,
including the entire Property (504 acres) and the Adjacent Property (408 acres). The Leasehold
Estate granted to Mineral Lessee by the Lease includes a fee simple determinable interest in the
Mineral Estate, including the exclusive right to prospect, explore, test (including seismic and
geologic investigations), drill for, produce, mine, extract, transport and remove oil, natural gas,
casinghead gas, and other hydrocarbon substances from the Property and the Adjacent Property.
Pursuant to the Lease, Mineral Lessee has the right of ingress and egress to and from the surface
of the Property and the Adjacent Property as necessary (consistent with the terms and limitations
of the Lease and applicable law) to conduct prospecting, exploration, testing (including seismic
and geologic investigations), drilling for, producing, mining, extracting, transporting and
removing oil, natural gas, casinghead gas, and other hydrocarbon substances in, on or under the
Property and Adjacent Property. Said rights and activities are referred to herein as the
"Extraction Operations."
F. The Mineral Owners are the owners of the reversionary interest to the Mineral
Lessee's interest in the Mineral Estate and are the successors to the lessor of the Lease.
G. The Mineral Estate and the Leasehold Estate are subject to that certain Unit
Agreement, Rio Vista Gas Unit, Contra Costa, Sacramento and Solano Counties, California,
dated June 3, 1964, and recorded July 9, 1964, at Book 1280, Page 451, Official Records of
Solano County (the "Unit Agreement"). Mineral Owners are parties to the Unit Agreement and
receive a share of royalties for gas produced within the lands covered by the Unit Agreement,
said lands defined as the "Unit Area" in the Unit Agreement. Mineral Lessee is the current Unit
Operator, as defined in the Unit Agreement, and the current sole Working Interest Owner, as
defined in the Unit Agreement. Mineral Lessee operates numerous existing natural gas wells in
arid below the Unit Area, and has the right under the Unit Agreement to conduct Extraction
Operations within the Unit Area. Mineral Owners and Mineral Lessee's predecessors-in-interest
entered into the Unit Agreement. Mineral Lessee has rights under the Unit Agreement, and
various other leases, to explore, develop and extract natural gas, condensate and associated
hydrocarbons within the Unitized Formation of the Unit Area as defined in the Unit Agreement,
as well as the right of ingress and egress to and from the surface of the Unit Area as provided in
the Unit Agreement and relevant leasehold agreements.
H. As of the Effective Date, Mineral Lessee has fourteen (14) existing gas well sites
on the Property (each containing a well) and plans for six (6) additional well sites on the
Property (these twenty (20) well sites are referred to collectively as the "Well Sites"). A Well
Sites Plan shows the locations of all twenty (20) Well Sites, and is attached as Exhibit E . For
purposes of this Easement Deed, each of the Well Sites is a "Well Site." The Well Sites will be
used to support existing wells, and to drill new wells. Pipelines presently exist on the Property to
support Mineral Lessee's existing Well Sites, and Mineral Lessee needs additional new pipelines
on the Property to support the Well Sites (the pipelines presently existing on the Property and
new pipelines planned by Mineral Lessee on the Property are referred to collectively as the
"Pipelines"). Pursuant to the Lease and the Unit Agreement, Mineral Lessee also has numerous
existing gas wells and pipelines located on the Adjacent Property, and has plans for additional
wells and pipelines on the Adjacent Property.
L Mineral Lessee is required to obtain certain approvals and/or permits ("City
Extraction Approvals") from the City for the development of new natural gas facilities, including
new wells and redrilling and deepening existing wells, pursuant to Chapter 13.12 of the City's
Municipal Code ("Natural Gas Ordinance" or "NGO"). Mineral Lessee may also need to obtain
approvals from the California Department of Conservation, Division of Oil, Gas and Geothermal
Resources and possibly from other agencies such as the U.S. Fish and Wildlife Service and the
California Department of Fish and Game for these drilling activities within the Well Sites
("Other Agency Extraction Approvals"). As used in this Easement Deed, the terms City
Extraction Approvals and Other Agency Extraction Approvals shall concern only such drilling
activities within the Property (and not drilling activities within the Adjacent Property for which
Mineral Lessee is required to seek separate approvals).
J. In conjunction with the development of the surface of the Property for activities,
structures and uses that are not related to mineral extraction ("Surface Development"), Owners
are required to obtain approvals and/or permits from the City (including a subdivision map,
pursuant to California Government Code Section 66410 et seq.), and possibly other
governmental agencies, for Surface Development and related activities, construction and
operations (collectively "Surface Development Approvals").
K. As a general matter, and subject to the terms and limitations of the Lease and
applicable law, Mineral Lessee currently has non-exclusive rights to use the surface of the
Property for the purposes set forth in the Lease and the Unit Agreement. Absent an agreement to
specify which portion(s) of the Property can be used by which Party for what purpose(s), a
possibility exists for conflict between and among the Parties as each exercises its rights in the
Property. Accordingly, Mineral Owners and Mineral Lessee desired assurances and certainty
that they will be able to continue existing Extraction Operations on certain portions of the
Property, and in and below the Unit Area, under the terms and conditions of the Lease and the
Unit Agreement and will be able to develop new Extraction Operations within the Well Sites,
and in and below the Unit Area, under the terms and conditions of the Lease and the Unit
Agreement. Similarly, Owners desire assurances and certainty that the Extraction Operations on
the Property will be confined to certain areas of the Property, so that Owners can proceed with
Surface Development.
L. The Parties 5 desired assurances and certainties are set forth in that certain
Accommodation Agreement - Del Rio Hills ("Accommodation Agreement"), recorded on
at Series # in the Official Records of Solano County.
M. Among other matters, the Accommodation Agreement (at Section 6.h) granted
Mineral Owners and Mineral Lessee a temporary, unrestricted easement (the "Temporary Floating
Pipeline/Utility Easement") over all of the Property for purposes of installing, operating and
maintaining pipelines and related appurtenances necessary and convenient for Mineral Lessee's
operations within the Property, the Unit Area and elsewhere. The Temporary Floating
Pipeline/Utility Easement arose automatically and without the need for further documentation or
approval upon the Parties' execution of the Accommodation Agreement; the Parties covenanted
and agreed that the recordation of the Accommodation Agreement constituted sufficient and
appropriate evidence of the grant of the Temporary Floating Pipeline/Utility Easement. The
Temporary Floating Pipeline/Utility Easement will continue in full force and effect until such
time as one or more final subdivision maps are recorded for Surface Development. Upon
recordation of a final subdivision map for Surface Development, the Temporary Floating
Pipeline/Utility Easement for only the portion of the Property which is the subject of the final
subdivision map will automatically terminate and be of no further force or effect. The
Temporary Floating Pipeline/Utility Easement for those portions of the Property which are not
the subject of a final subdivision map for Surface Development will continue in full force and
effect. Notwithstanding said automatic termination, Mineral Lessee and Mineral Owners will
execute and deliver an appropriate instrument or document manifesting release of all or portions,
as applicable, of the Temporary Floating Pipeline/Utility Easement following Owner's delivery
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of (i) a written request for such instrument or document, and (ii) a copy of the recorded final
subdivision map for Surface Development of the Property.
N. Pursuant to the Accommodation Agreement, Mineral Owners and Mineral Lessee
desire to obtain exclusive easements across certain portions of the Property in order to establish,
develop, maintain and repair and access Well Sites and to conduct Extraction Operations.
AGREEMENT :
In consideration of the mutual promises contained in this Easement Deed and of other
valuable consideration, the receipt and sufficiency of which the Parties expressly acknowledge, it is
agreed as follows:
1. Grant of Pipeline and Utility Easements .
Grantors hereby gratnt to Grantees temporary and permanent nonexclusive
easements that are reasonably necessary for: (i) all pipelines and utilities necessary to service
each Well Site, including without limitation, installation of conduits, pipe in pipe and wire in
pipe; (ii) all Well Site flow lines and utility lines inside of protective conduit; (iii) pipeline stub-
outs and utility or pipeline connections; and (iv) ingress and egress over and under the easement
area for the installation, construction, operation, repair, maintenance, relocation and replacement
of all pipelines and utilities (the "Pipeline/Utility Easements"). The Pipeline/Utility Easements
shall be in gross and shall benefit Mineral Lessee and Mineral Owners, and are hereby conveyed
to Mineral Owners and Mineral Lessee for non-exclusive use by Mineral Owners and Mineral
Lessee. The Pipeline/Utility Easements as shown on the Well Sites Plan shows the Parties'
agreed upon location of temporary and permanent easements which shall consist of (i) a
permanent easement area a minimum of twenty (20) feet wide; and (ii) a temporary construction
easement area to be used by Mineral Lessee during construction of pipelines and access
roadways of an additional fifteen (15) feet on both sides of the twenty (20) foot permanent
easement. The Pipeline/Utility Easements are designed to ensure, and shall have a scope
sufficient to ensure, that any gas produced from any location (whether or not from below the
Property, the Unit Area or elsewhere) that flows into the pipeline system on the Property has at
least two outlets to exit the system on the Property.
2. Pipeline and Utility Construction .
a. If Mineral Lessee Constructs its Improvements in an Area Before Owners
Construct their Improvements in the Same Area .
At all times, but prior to Surface Development in the vicinity of the area of the
Property where Mineral Lessee desires to construct and install a pipeline or other utilities,
Mineral Lessee shall be responsible for the installation and cost of all improvements necessary to
transport production from its wells and to support its Well Sites with utility service. Mineral
Lessee shall construct such improvements in the location of the Pipeline/Utility Easements set
forth on the Well Sites Plan. Owners shall be entirely responsible to ensure that Mineral
Lessee's improvements are not disturbed by any subsequent improvements, including grading, of
the surface by Owners. In the event that Owners and Mineral Lessee agree, after Mineral Lessee
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has installed pipelines and utilities, to modify the locations of any Pipeline/Utility Easements to
accommodate Owners' Surface Development, then Owners shall be responsible for all costs of
any resulting relocation of the pipelines and utilities. Mineral Lessee shall have the right to
approve the time and manner of any relocation of pipelines or utilities done by Owners, or at
Owners' request, prior to such relocation; such approval shall not be unreasonably withheld,
conditioned or delayed, provided such relocation will not result in any substantial or material
interruption of pipeline or utility service to Mineral Lessee's Extraction Operations on the
Property. For purposes of this Easement Deed, "substantial or material interruption" shall mean
a complete shut down, for a period exceeding forty-eight (48) consecutive hours, of one or more
of Mineral Lessee's actively producing gas wells which utilized the affected Pipeline/Utility
Easements, in spite of Mineral Lessee's commercially reasonable efforts to re-route gas
production from the affected gas well(s) using other existing pipelines. Mineral Lessee shall
issue its approval or disapproval within thirty (30) days of receipt of Owner's plans and
specifications for the relocation of any pipelines or utilities. Mineral Lessee acknowledges and
agrees that there will likely be temporary disruption of pipeline or utility service to the
Extraction Operations on the Property when the new pipelines and utility connections are made.
In the event that any such relocation activities result in a substantial or material interruption,
Owner shall pay to Mineral Lessee a fee equal to Mineral Lessee's lost production value from
the affected gas well(s) for the period (i) beginning at the expiration of the initial forty-eight (48)
consecutive hours, and (ii) ending when the pipeline connection is fully restored to the actively
producing gas well(s). For purposes of this Section 2. a, the fee payable to Mineral Lessee for the
lost production value of an affected gas well shall be determined by the average of the affected
gas well's daily yield for the thirty (30) day period preceding the date of the disruption, and
based upon the contract price Mineral Lessee is then receiving from the purchaser of the gas
well's production.
b. If Owners Construct their Improvements in an Area before Mineral Lessee
Constructs its Improvements in the Same Area .
At all times after Owners have commenced development of the Property
consistent with the Accommodation Agreement and the Surface Development Approvals on or
near any of Mineral Lessee's Pipeline/Utility Easements, Owners shall install improvements
(such as pipelines, water lines, telemetry/telephone lines and electrical lines) within the
Pipeline/Utility Easements according to the Well Sites Plan or Survey, as appropriate (see )
Section 1 .f of the Accommodation Agreement), to service existing or future wells at the Property
as shown on the Well Sites Plan or Survey, as appropriate. Owners shall be responsible for the
construction costs and installation of such pipelines and utilities; Mineral Lessee shall be
responsible for the materials costs of the pipeline and other utility materials. The intent of this
provision is to allow efficiencies of constructing Mineral Lessee's pipelines and other
underground utilities at the same time Owners are building streets, sidewalks, and other
infrastructure improvements. Prior to construction, Owners shall submit plans for such pipelines
and other utility improvements to Mineral Lessee for Mineral Lessee's review and approval to
ensure that the pipelines and improvements meet or comply with Mineral Lessee's specifications
and standards and/or requirements of any approval or permit granted to Mineral Lessee for
Extraction Operations. Mineral Lessee shall not unreasonably withhold, condition or delay
approval of Owners' plans for such pipelines and other utility improvements, and Mineral Lessee
shall issue its written approval or disapproval within thirty (30) days of receipt of Owners' plans
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and specifications. If Mineral Lessee disapproves of the proposed plans, Mineral Lessee's
written notice to Owners shall specify the nature of Mineral Lessee's objections and identify
proposed revisions to the plans to correct the objections. Within thirty (30) days following
receipt of Mineral Lessee's disapproval notice and proposed revisions, Owners shall notify
Mineral Lessee in writing concerning whether Owners accept or reject the proposed revisions. If
Owners accept the proposed revisions, the plans shall be duly modified and implementation of
the pipeline improvements shall proceed accordingly. If Owners reject the proposed revisions,
Owners and Mineral Lessee shall resolve their outstanding issues in accordance with the dispute
resolution provisions set forth in Sections 12.a and 12.b of the Accommodation Agreement.
[This reference to the AA may work, as it relates to the construction phase, when A A still on title,
prior to any conveyances of the areas burdened by the easements (open space, roads, etc.) to the
City.]
3. Pipeline and Utility Relocation .
a. Pipeline Location . Prior to the recordation of a final subdivision map for
Surface Development, Mineral Lessee shall have the right to locate pipelines and utilities under
the Temporary Floating Pipeline/Utility Easement, as granted by the Accommodation Agreement
and described in Recital M of this Easement Deed. The final location of the Pipeline/Utility
Easements shall be determined by those certain City Extraction Approvals, a development
agreement (with a term of at least fifteen (15) years) between Mineral Lessee and the City,
pursuant to California Government Code Section 65864 et seq., those Other Agency Extraction
Approvals, and the development agreement with the City vesting those extraction approvals
("Development Agreement"), with potential modification as set forth herein to accommodate
Owners' Surface Development.
b. Relocation of Existing Pipelines . The location of some of the
Pipeline/Utility Easements may need to change from time to time as Owners obtain approvals
and/or permits from the City and other governmental agencies for Surface Development over
time. Owners may, at any time, request that certain existing (as of the Effective Date) utilities
and pipelines located within the Property ("Existing Utilities") that conflict with any of Owners'
Surface Development Approvals be abandoned and, as appropriate, removed by Owners. These
utilities and pipelines may be owned by Mineral Lessee or by other parties such as Pacific Gas
and Electric Company ("PG&E") (utilities and pipelines owned by PT&E or other third parties
are referred to herein as "Third Party Utilities"); Mineral Lessee may have the right to use Third
Party Utilities. Prior to abandonment and removal of any Existing Utilities, Owners must obtain
the written consent of the owner of the Existing Utility and, in the instance of Third Party
Utilities, of Mineral Lessee. If Mineral Lessee, and, if applicable, the owner of a Third Party
Utility, agree to the abandonment and removal, then Mineral Lessee shall not be responsible for
the abandonment and removal or any costs of same, and such costs shall be paid by Owners
and/or, if applicable, the owner of a Third Party Utility, as agreed between those parties. In the
event any of Mineral Lessee's pipelines or utilities need to be relocated and abandoned, such
pipeline shall not be abandoned until a replacement pipeline or utility of sufficient size, quality
and location has been installed and commissioned (if deemed necessary by Mineral Lessee) to
transport gas produced from any location (whether or not from below the Property, the Unit Area
or elsewhere) that flows into the pipeline system on the Property.
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c. Relocation of Pipelines or Utilities After Completion of Surface
Development , At all times after Owners have completed Surface Development of the Property,
any pipelines or utility improvements that Mineral Lessee needs to replace, repair or improve
(presuming such need was not caused by Owners, in which case Owners shall be entirely
responsible for the costs to replace, repair or improve) shall be done at Mineral Lessee's sole
cost. Where there are surface improvements in the area of the Pipeline/Utility Easements,
including without limitation, landscaping, Mineral Lessee shall be responsible for all incremental
costs associated with construction conflicts caused by such improvements and Mineral Lessee
shall be responsible for restoring the surface to a condition comparable to that which existed
prior to Mineral Lessee's construction activities and for replacing any surface improvements
damaged by Mineral Lessee's activities.
4. Environmental Indemnification .
With regard to their respective activities on the Property as permitted by this
Easement Deed, the Parties shall comply with all environmental laws, statutes and regulations
issued by federal, state and local governmental agencies ("Agencies") and shall conduct their
activities on the Property according to environmental standards set by the Agencies, including
without limitation, hazardous waste, toxic substances, hydrocarbons and petroleum based
substances, water, wetlands, endangered and threatened species and air standards ("Agency
Environmental Standards"). The Parties Lessee shall also comply with California Health and
Safety Code Section 25359.7. If a Party's use of or activity on the Property results in a loss to
any other Party due to a violation of Agency Environmental Standards resulting from use of the
Property, whether an enforcement action is brought by an agency or a private citizen, the Party
whose use caused the loss shall defend (using legal counsel reasonably satisfactory to the other
Party), indemnify and hold harmless the other Party(ies) from the loss and any resulting penalties
and fines; and the Party whose use led to the violation shall, at its sole cost, respond to all such
violations for its benefit and for the benefit of the other Party(ies), including all clean-up,
demolition, detoxification, and disposal and the preparation of any closure or other required
plans. In the case of joint or contributory negligence, breach of contract or other fault or strict
liability on the part of the Party seeking indemnification, principles of comparative negligence
shall be followed and each Party shall bear the proportionate cost of any loss, damage, expense,
or liability attributable to such Party's negligence, breach of contract, use or other fault or strict
liability. This Section 4 applies only to activities and uses occurring after the Effective Date.
There are no third party beneficiaries to this Section 4. The environmental indemnification
obligations contained in this Section 4 shall survive the termination of each Parties' interest in
the Property and the Pipeline/Utility Easements.
5. Termination of the Pipeline/Utility Easements .
a. Mineral Lessee's interest in the Pipeline/Utility Easements shall terminate
when Mineral Lessee releases the Pipeline/Utility Easements to Owners and Mineral Owners.
Mineral Lessee shall have the right, in its sole discretion, to elect to abandon any pipeline in
place. Upon expiration or termination of its interest in the Pipeline/Utility Easements, Mineral
Lessee or its successor, shall promptly and expeditiously execute and acknowledge, on request of
Owners, Mineral Owners or their title insurer, a release to Mineral Owners formally releasing of
record Mineral Lessee's interests in, and rights under, the Pipeline/Utility Easements.
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b. Mineral Owners' interest in the Pipeline/Utility Easements shall terminate
at the earlier of (i) when the pipelines are not used to transport hydrocarbons for a period of
longer than one hundred eighty (180) days after Mineral Lessee has recorded a release of the
Pipeline/Utility Easements, (ii) when the ownership of the Mineral Estate and Surface Estate
merge, or (iii) when the Mineral Owners' interest in the Pipeline/Utility Easements is otherwise
released.
6. Binding Effect ,
The provisions of this Easement Deed shall be binding upon and inure to the
benefit of the Parties, their respective heirs, transferees and successors in interest, including,
without limitation, all subsequent owners of the Sale Property and the Retained Property, and the
burden of the Pipeline/Utilities Easements shall be covenants running with the land of the
Property. For purposes of apportioning responsibility and liability for the obligations generally
ascribed to "Owner" in this Easement Deed, and unless the context of this Easement Deed
otherwise specifically provides, (i) the fee estate owner of the Sale Property shall be responsible
and liable only for those obligations, actions and activities arising under this Easement Deed that
pertain to or otherwise stem from the ownership of the Sale Property; and (ii) the fee estate
owner of the Retained Property shall be responsible and liable only for those obligations, actions
and activities arising under this Easement Deed that pertain to or otherwise stem from the
ownership of the Retained Property. If the Option Agreement terminates or is terminated prior to
RVHHC acquiring any portion of the Property, the term RVHHC shall mean Surface Owners or
future developers taking their interest through Surface Owners, and RVHHC shall not have any
further rights under this Easement Deed. Mineral Lessee shall have the right to assign this
Easement Deed and any of Mineral Lessee's rights hereunder.
7. Mineral Lessee's Right to Authorize Use by Unit Operator; Mineral Lessee and
Mineral Owners' Right to Authorize Use by Third Party Utility Providers .
Mineral Lessee shall have the right to authorize the use of the Pipeline/Utility
Easements by the Unit Operator for the purposes of conducting Unit operations, so long as the
Unit Agreement is in effect. Mineral Lessee and Mineral Owner shall have the right to authorize
the use of the Pipeline/Utility Easements by any public or private utility company providing any
utilities serving the Well Sites, or as necessary to transport gas to market.
7. Status of Title .
This Easement Deed is made subject to all conditions, covenants, restrictions,
leases, easements, licenses, liens, encumbrances and claims of title of record which may affect
the Pipeline/Utility Easements.
8. Notices .
All notices, demands, or other communications required or permitted by this
Easement Deed or by law to be served on or given to a Party shall be in writing and shall be
deemed delivered (i) if personally delivered, upon delivery to the Party to whom directed, (ii) if
mailed, upon the expiration of forty-eight (48) hours from the date of mailing in the United
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States mail, registered or certified, return receipt requested, addressed to the Party address shown
below, or (iii) if faxed, upon receipt of the fax transmittal, transmitted to the Party fax number
shown below, provided the machine sending the fax provides a written confirmation of receipt:
If to the Surface Owners, at:
And to:
And to:
With a copy to:
And with a copy to:
If to Mineral Owners, at:
And to:
And to:
c/o Mr. John Wyro
The Wyro Company
40 Valley Drive
Orinda,CA 94563
Telephone: 925-254-5246
Fax: 925-254-5299
Richard and Sue Esperson
398 Crescent Drive
Rio Vista, CA 94571
Mrs. Jean Grimm
35 San Gabriel Dr.
Fairfax, CA 94930
Teri A. Bjorn, Of Counsel
Kronick, Moskovitz, Tiedemann & Girard
1675 Chester Avenue, Suite 320
Bakersfield, CA 93301
Telephone: 661-864-3800
Fax: 661-864-3810
David G. Kenyon, Esq.
7200 Redwood Blvd., Suite 404
Novato,CA 94945
Telephone: 415-892-1868
Fax: 415-892-1716
c/o Mr. John Wyro
The Wyro Company
40 Valley Drive
Orinda,CA 94563
Telephone: 925-254-5246
Fax; 925-254-5299
Richard and Sue Esperson
398 Crescent Drive
Rio Vista, CA 94571
Mrs. Jean Grimm
35 San Gabriel Dr.
Fairfax, CA 94930
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With a copy to:
And with a copy to: .
If to Mineral Lessee, at:
With a copy to:
And with a copy to:
If to the RVHHC, at:
And to:
teri A. Bjorn, of Counsel
Kronick, Moskovitz, Tiedemann & Girard
1675 Chester Avenue, Suite 320
Bakersfield, CA 93301
Telephone: 661-864-3800
Fax: 661-864-3810
David G. Kenyon, Esq.
7200 Redwood Blvd., Suite 404
Novato, CA 94945
Telephone: 415-892-1868
Fax: 415-892-1716
Rosetta Resources Operating, L.P.
1200 17th Street, Suite 770
Denver, CO 80202
Telephone: (720) 946-1315
Fax:(720)359-9140
Rosetta Resources, Inc.
Attn: General Counsel
717 Texas Avenue, Suite 2800
Houston, TX 77002
Telephone: (713) 335-4017
Fax: (7 13) 335-4136
Stephen R. Finn, Esq.
One Market Street, Spear Tower
San Francisco, CA 94105
Telephone: 415-442-1251
Fax: 415-442-1001
Rio Vista Hills Holding Company, LLC
c/o Lewis Operating Corp.
9216Kiefer Blvd, Suite 8
Sacramento, CA 95827
Attn: Douglas Mull, Vice Pres.
Telephone: 916-363-2617 ext.226
Fax: 916-364-9353
Lewis Operating Corp.
Legal Department
1 1 56 N. Mountain Avenue
Upland, CA 91786
Attn: W. Bradford Francke, Esq.
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Telephone: 909-946-7538
Fax: 909-946-6725
With a copy to: Law Offices of Gregory D. Thatch
1730 T* Street, Suite 220
Sacramento, C A 95814
Attn: Michael Devereaux, Esq.
Telephone: 916-443-6956.
Fax: 916-443-4632
9. Choice of Law; Venue .
This Easement Deed shall be governed by and construed in accordance with the
substantive and procedural laws of the State of California, excluding any laws that require the
application of another jurisdiction's laws. This Easement Deed is entered into and is to be
performed in Solano County, California, arid accordingly the only appropriate venue for a
dispute under this Easement Deed is in the Solano County Superior Court of California. All
references to codes in this Easement Deed are to California or City codes, unless otherwise
specified.
10. Entire Agreement .
With the exception of the Lease, Unit Agreement, Accommodation Agreement
and Declaration of Covenants, Conditions and Restrictions for the Del Rio Hills Project, this
Easement Deed (including the Exhibits to this Easement Deed) supersedes any and all other
agreements, either oral or in writing, between the parties with respect to the subject matter and
contains all of the covenants and agreements between the parties with respect to such matter, and
each party to this Easement Deed acknowledges that no representations, inducements, promises
or agreements, oral or otherwise, have been made by any party, or anyone acting on behalf of
any party, which are not embodied herein, and that no other agreement, statement or promise not
contained in, or contemplated by this Easement Deed shall be valid or binding.
11. Modification .
This Easement Deed may be modified or amended only by a written recordable
document signed and acknowledged by the (a) then-current Surface Owners, (b) RVHHC or its
successor, if any, (c) then-current Mineral Lessee, and (d) then-current Mineral Owners, or by their
respective successors-in-interest, transferees or assigns.
12. Partial Invalidity .
In the event a court of law determines that any provision of this Easement Deed, or
portion thereof, is prohibited, unlawful, or unenforceable under any applicable law of any
jurisdiction, the remainder of the provisions hereof shall remain in full force and effect and shall
in no way be affected, impaired or invalidated.
- 13-
13. Time .
Time is of the essence in the performance of the parties* respective obligations
contained in this Easement Deed.
14. Counterparts .
This Easement Deed may be executed in any number of counterparts all of which
when executed shall constitute one document.
IN WITNESS WHEREOF, Grantors and Grantees have executed this Easement Deed as of
the Effective Date.
GRANTORS:
THE GRIMM-RIO VISTA FAMILY
LIMITED PARTNERSHIP
By_
General Partner
- 14-
RICHARD W. ESPERSON also known as
Richard W. Esperson, Jr., as Trustee of the
Richard W. Esperson and Irene Sue
Esperson Family Trust dated October 17,
1991
IRENE S. ESPERSON, as Trustee of the
Richard W. Esperson and Irene Sue
Esperson Family Trust dated October 17,
1991
MARK ESPERSON
GARY ESPERSON
STEPHEN ESPERSON
KIMBERLY ESPERSON
RIO VISTA HILLS HOLDING
COMPANY, LLC
a Delaware limited liability company
By North Mountain Corporation,
a California corporation
Its Sole Manager
By
By.
GRANTEES:
NORMA JEAN GRIMM, also known as
Jean Harris Grimm, as Trustee of the Grimm
Family Trust dated October 4, 1990 -
Survivors Trust
RICHARD W. ESPERSON, JR.
JOAN ESPERSON WEDDELL
DAVID SANTOS
RICHARD SANTOS
STEPHEN ESPERSON
GARY ESPERSON
MARK ESPERSON
SUSAN A. BORGESEN, formerly Susan A.
Woodworth
SANDRA J. DICKSON, formerly Sandra
Grimm
SHARON E. HARRIS, formerly Sharon
Grimm
STEPHEN A. GRIMM, formerly Stephen
Grimm
THE GRIMM-RIO VISTA FAMILY
LIMITED PARTNERSHIP
By
General Partner
JEAN HARRIS GRIMM
DAVID L. SANTOS, as Surviving Trustee
of the David L. and Laura E. Santos
Revocable Trust dated February 12, 2002
RICHARD W. ESPERSON, also known as
Richard W. Esperson, Jr., as Trustee of the
Richard W. Esperson and Irene Sue
Esperson Family Trust dated October 17,
1991
IRENE S. ESPERSON, as Trustee of the
Richard W. Esperson and Irene Sue
Esperson Family Trust dated October 17,
1991
ROSETTA RESOURCES OPERATING,
L.P., a California limited partnership
By :
By.
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